Modified Bouganin Proteins, Cytotoxins and Methods and Uses Thereof

ABSTRACT

The invention provides modified forms of bouganin protein having biological activity and a reduced propensity to activate human T cells as compared to the non-modified bouganin protein. The invention also provides T-cell epitope peptides of bouganin, and modified T-cell epitope peptides of bouganin which have a reduced propensity to activate human T cells as compared to the non-modified T-cell epitope peptide. The invention also provides cytotoxins having the having a ligand that binds to a cancer cells attached to the modified bouganin proteins. Also provided are methods of inhibiting or destroying mammalian cancer cells using the cytotoxins of the invention and pharmaceutical compositions for treating human cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC 119(e) from U.S.provisional application No. 60/554,580, filed on Mar. 19, 2004 and U.S.provisional application No. 60/630,571, filed on Nov. 26, 2004, whichare incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The invention relates to modified bouganin proteins and cytotoxinscontaining the modified proteins useful as therapeutics against cancer.Specifically, T-cell epitopes are removed or altered to reduceimmunogenicity of the bouganin toxins.

BACKGROUND OF THE INVENTION

There are many instances whereby the efficacy of a therapeutic proteinis limited by an unwanted immune reaction to the therapeutic protein.Several mouse monoclonal antibodies have shown promise as therapies in anumber of human disease settings but in certain cases have failed due tothe induction of significant degrees of a human anti-murine antibody(HAMA) response [Schroff, R. W. et al (1985) Cancer Res. 45: 879-885;Shawler, D. L. et al (1985) J. Immunol. 135: 1530-1535]. For monoclonalantibodies, a number of techniques have been developed in attempt toreduce the HAMA response [WO 89/09622; EP 0239400; EP 0438310; WO91/06667]. These recombinant DNA approaches have generally reduced themouse genetic information in the final antibody construct whilstincreasing the human genetic information in the final construct.Notwithstanding, the resultant “humanised” antibodies have, in severalcases, still elicited an immune response in patients [Issacs J. D.(1990) Sem. Immunol. 2: 449, 456; Rebello, P. R. et al (1999)Transplantation 68: 1417-1420].

The key to the induction of an immune response is the presence withinthe protein of peptides that can stimulate the activity of T-cells viapresentation on MHC class II molecules, so-called “T-cell epitopes”.Such T-cell epitopes are commonly defined as any amino acid residuesequence with the ability to bind to MHC Class II molecules. Implicitly,a “T-cell epitope” means an epitope which when bound to MHC moleculescan be recognized by a T-cell receptor (TCR), and which can, at least inprinciple, cause the activation of these T-cells by engaging a TCR topromote a T-cell response.

MHC Class II molecules are a group of highly polymorphic proteins whichplay a central role in helper T-cell selection and activation. The humanleukocyte antigen group DR (HLA-DR) are the predominant isotype of thisgroup of proteins; however, isotypes HLA-DQ and HLA-DP perform similarfunctions. In the human population, individuals bear two to four DRalleles, two DQ and two DP alleles. The structure of a number of DRmolecules has been solved and these appear as an open-ended peptidebinding groove with a number of hydrophobic pockets which engagehydrophobic residues (pocket residues) of the peptide [Brown et al(1993) Nature 364: 33; Stern et al (1994) Nature 368: 215]. Polymorphismidentifying the different allotypes of class II molecule contributes toa wide diversity of different binding surfaces for peptides within thepeptide binding groove and at the population level ensures maximalflexibility with regard to the ability to recognize foreign proteins andmount an immune response to pathogenic organisms.

An immune response to a therapeutic protein proceeds via the MHC classII peptide presentation pathway. Here exogenous proteins are engulfedand processed for presentation in association with MHC class IImolecules of the DR, DQ or DP type. MHC Class II molecules are expressedby professional antigen presenting cells (APCs), such as macrophages anddendritic cells amongst others. Engagement of a MHC class II peptidecomplex by a cognate T-cell receptor on the surface of the T-cell,together with the cross-binding of certain other co-receptors such asthe CD4 molecule, can induce an activated state within the T-cell.Activation leads to the release of cytokines further activating otherlymphocytes such as B cells to produce antibodies or activating T-killercells as a full cellular immune response.

T-cell epitope identification is the first step to epitope eliminationas recognized in WO98/52976; WO00/34317; WO02/069232; WO02/079232; andWO02/079415. In these teachings, predicted T-cell epitopes are removedby the use of judicious amino acid substitution within the protein ofinterest. Besides computational techniques, there are in vitro methodsfor measuring the ability of synthetic peptides to bind MHC class IImolecules. An exemplary method uses B-cell lines of defined MHC allotypeas a source of MHC class II binding surface and may be applied to MHCclass II ligand identification [Marshall K. W. et al. (1994) J. Immunol.152:4946-4956; O'Sullivan et al (1990) J. Immunol. 145: 1799-1808;Robadey C. et al (1997) J. Immunol 159: 3238-3246]. However, suchtechniques are not adapted for the screening of multiple potentialepitopes to a wide diversity of MHC allotypes, nor can they confirm theability of a binding peptide to function as a T-cell epitope.

Techniques exploiting soluble complexes of recombinant MHC molecules incombination with synthetic peptides have also come into use [Kern, F. etal (1998) Nature Medicine 4:975-978; Kwok, W. W. et al (2001) TRENDS inImmunol. 22:583-588]. These reagents and procedures are used to identifythe presence of T-cell clones from peripheral blood samples from humanor experimental animal subjects that are able to bind particularMHC-peptide complexes and are not adapted for screening multiplepotential epitopes to a wide diversity of MHC allotypes.

Biological assays of T-cell activation offer a practical option toproviding a reading of the ability of a test peptide/protein sequence toevoke an immune response. Examples of this kind of approach include thework of Petra et al using T-cell proliferation assays to the bacterialprotein staphylokinase, followed by epitope mapping using syntheticpeptides to stimulate T-cell lines [Petra, A. M. et al (2002) J.Immunol. 168: 155-161]. Similarly, T-cell proliferation assays usingsynthetic peptides of the tetanus toxin protein have resulted indefinition of immunodominant epitope regions of the toxin [Reece J. C.et al (1993) J. Immunol. 151: 6175-6184]. WO99/53038 discloses anapproach whereby T-cell epitopes in a test protein may be determinedusing isolated sub-sets of human immune cells, promoting theirdifferentiation in vitro and culture of the cells in the presence ofsynthetic peptides of interest and measurement of any inducedproliferation in the cultured T-cells. The same technique is alsodescribed by Stickler et al. [Stickler, M. M. et al (2000) J.Immunotherapy 23:654-660], where in both instances the method is appliedto the detection of T-cell epitopes within bacterial subtilisin. Such atechnique requires careful application of cell isolation techniques andcell culture with multiple cytokine supplements to obtain the desiredimmune cell sub-sets (dendritic cells, CD4+ and or CD8+ T-cells) and isnot conducive to rapid through-put screening using multiple donorsamples.

Recently a combination approach using population based T-cellproliferation assays and in silico simulation of peptide MHC binding inthe design of epitope depleted proteins has also been advanced [WO03/104803].

As depicted above and as consequence thereof, it would be desirable toidentify and to remove or at least to reduce T-cell epitopes from aprincipal therapeutically valuable but originally immunogenic peptide,polypeptide or protein.

SUMMARY OF THE INVENTION

The invention is conceived to overcome the practical reality thatsoluble proteins introduced with therapeutic intent in humans cantrigger an immune response resulting in development of host antibodiesthat bind to the soluble protein. The present invention seeks to addressthis by providing bouganin proteins with reduced propensity to elicit animmune response. According to the methods described herein, theinventors have identified the regions of the bouganin moleculecomprising the critical T-cell epitopes driving the immune responses tothis protein.

The present invention relates to a modified bouganin protein wherein themodified bouganin has a reduced propensity to elicit an immune response.In a preferred embodiment, the modified bouganin has a reducedpropensity to activate T-cells and the modified bouganin is modified atone or more amino acid residues in a T-cell epitope. The T-cell epitopesare selected preferably from the group consisting of:

a) AKVDRKDLELGVYKL, (epitope region R1, SEQ ID NO: 2) b)LGVYKLEFSIEAIHG; (epitope region R2, SEQ ID NO: 3) and c)NGQEIAKFFLIVIQM. (epitope region R3, SEQ ID NO: 4)

The present invention also relates to a cytotoxin comprising a targetingmoiety attached to a modified bouganin protein of the invention. In oneembodiment, the targeting moiety is a ligand that binds to a cancercell. In a further embodiment, the ligand is an antibody or antibodyfragment that binds to a cancer cell. In a particular embodiment, theantibody recognizes Ep-CAM or tumor-associated antigen. In a mostparticular embodiment, the present invention provides a cytotoxincomprising VB6-845 or VB6-011.

In another aspect, the invention provides a method of inhibiting ordestroying cancer cells comprising administering a cytotoxin of theinvention to the cancer cells.

The present invention also relates to a method of treating cancer byadministering a cytotoxin of the invention to an animal in need thereof.

Still further, a process is provided for preparing a pharmaceutical fortreating an animal with cancer comprising the steps of identifyingT-cell epitopes of bouganin, modifying one or more amino acid residuesin a T-cell epitope to prepare a modified bouganin having reducedpropensity to activate T-cells; preparing a cytotoxin have acancer-binding ligand attached to a modified bouganin; and suspendingthe cytotoxin in a pharmaceutically acceptable carrier, diluent orexcipient.

In a further aspect, the invention provides a pharmaceutical compositionfor treating an animal with cancer comprising the cytotoxin of theinvention and a pharmaceutically acceptable carrier, diluent orexcipient.

The cytotoxins, compositions and methods of the present invention may beused to treat various forms of cancer such as colorectal cancer, breastcancer, ovarian cancer, pancreatic cancer, head and neck cancer, bladdercancer, gastrointestinal cancer, prostate cancer, small cell and nonsmall cell lung cancer, sarcomas, gliomas, T- and B-cell lymphomas.

The invention also provides the T-cell epitope peptides of the bouganinprotein and the modified T-cell epitope peptides of the invention.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 shows results of activity assays of the T-cell epitope depletedmodified bouganin proteins Bou156 (panel A) and Bou157 (panel B). Bou156comprises the substitutions V123A, D127A, Y133N and 1152A. Bou157comprises the substitutions V123A, D127A, Y133Q and 1152A. Both assaysets are conducted using wild type protein and a disabled modifiedbouganin (Y70A) as controls. Activity is expressed as % measuredluciferase activity versus concentration of bouganin protein in theassay.

FIG. 2 shows T-cell proliferation assay results for three syntheticpeptides and 2 different PBMC donor samples. The peptides designatedDel-41, Del-44 and Del-50 were tested at 1 μM final concentration (panelA) and 5 μM final concentration (panel B). These peptides are derivedfrom the immunogenic regions of the bouganin molecule and containsubstitutions designed to eliminate their immunogenicity.

FIG. 3 illustrates VB6-845, a modified bouganin cytotoxin having a Fabanti-Ep-CAM, wherein the de-bouganin (Bou156) is linked to theC-terminus of the CH domain via a furin linker. FIG. 3A illustrates thedicistronic unit encoding the pro-sequences, FIG. 3B illustrates thenucleic acid coding sequence (SEQ ID NO:15) and the amino acid sequence(SEQ ID NO:16) of the pro-sequences and FIG. 3C illustrates theassembled VB6-845 protein without the peIB sequences.

FIG. 4 illustrates the map of the expression vector plNG3302. Inserts ofthe examples were ligated in 3302 vector using EcoRI and XhoIrestriction sites.

FIG. 5 illustrates the control Fab anti-Ep-CAM construct without theplant toxin, de-bouganin (VB5-845). FIG. 5A illustrates the dicistronicunit encoding the pro-sequences, FIG. 5B illustrates the nucleic acidcoding sequence (SEQ ID NO:17) and the amino acid sequence (SEQ IDNO:18) of the pro-sequences and FIG. 5C illustrates the assembledVB5-845 protein without the peIB sequences.

FIG. 6 illustrates the Fab anti-Ep-CAM de-bouganin constructVB6-845-C_(L)-de-bouganin, wherein the Bou156 is linked at theC-terminus of the C_(L) domain. FIG. 6A illustrates the dicistronicunits encoding the pro-sequences, FIG. 6B illustrates the nucleic acidcoding sequence (SEQ ID NO:19) and the amino acid sequence (SEQ IDNO:20) of the pro-sequences and FIG. 6C illustrates the assembledVB6-845-C_(L)-de-bouganin protein without the peIB sequences.

FIG. 7 illustrates the Fab anti Ep-CAM, de-bouganin construct,VB6-845-NV_(H)-de-bouganin, wherein Bou156 is linked to the N-terminusof the V_(H) domain. FIG. 7A illustrates the dicistronic units encodingthe pro-sequences, FIG. 7B illustrates the nucleic acid coding sequence(SEQ ID NO:21) and the amino acid sequence (SEQ ID NO:22) of thepro-sequences and FIG. 7C illustrates the assembledVB6-845-NV_(H)-de-bouganin protein without the peIB sequences.

FIG. 8 illustrates the Fab anti-Ep-CAM de-bouganin construct,VB6-845-NV_(L)-de-bouganin, wherein Bou156 is linked to the N-terminusof the V_(L) domain. FIG. 8A illustrates the dicistronic units encodingthe pro-sequences, FIG. 8B illustrates the nucleic acid coding sequence(SEQ ID NO:23) and the amino acid sequence (SEQ ID NO:24) of thepro-sequences and FIG. 8C illustrates the assembledVB6-845-NV_(L)-de-bouganin protein without the peIB sequences.

FIG. 9 is a Western Blot illustrating the expression of VB6-845(construct of FIG. 3) and VB6-845-CL-de-bouganin (Bou156) (construct ofFIG. 6) in the supernatant of induced E104 cells at lab-scale.

FIG. 10 illustrates the results of the flow cytometry reactivitystudies. FIG. 10A illustrates the reactivity of VB6-845 (construct ofFIG. 3) and VB6-845-C_(L)-de-bouganin (construct of FIG. 6) inEp-CAM-positive cell lines CAL 27 and OVCAR-3 and Ep-CAM-negative cellline A-375, while FIG. 10 B, illustrates the results of the same testsconducted with VB6-845 (construct of FIG. 3) and VB6-845-gelonin(construct of FIG. 14C) and control (PBS).

FIG. 11 is a graph illustrating the results of the competitionassay-VB6-845 and Proxinium™ in NIH:OVCAR-3 cells and as described inExample 7.

FIG. 12 is a graph illustrating the results of the cell free assay ofExample 7.

FIG. 13 illustrates the results of the MTS cytotoxicity assay of Example8 comparing the cytoxocity of VB6-845 (construct of FIG. 3),VB6-845-CL-de-bouganin (construct of FIG. 6) and de-bouganin (Bou156) inCAL 27 (FIG. 13A) and NIH:OVCAR3 (FIG. 13B) cells.

FIGS. 14A and B illustrate the results of the MTS cytotoxicity assay ofExample 8 comparing the cytoxocity of VB6-845 (construct of FIG. 3),VB6-845-gelonin (construct of FIG. 14C) and gelonin in CAL 27 (FIG. 14A)and NIH:OVCAR3 (FIG. 14B) cells. FIG. 14C illustrates the nucleic acidcoding sequence (SEQ ID NO:25) and the amino acid sequence (SEQ IDNO:26) of the VB6-845-gelonin construct.

FIG. 15 illustrates the nucleic acid coding sequence (SEQ ID NO: 27) andthe amino acid sequence (SEQ ID NO:28) of the pro-sequences of VB6-011.

FIG. 16 illustrates the results of the MTS cytotoxicity assay of Example9 showing the cytotoxicity of VB6-011 in MB-435S cells.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have identified T-cell epitopes in bouganin, and havedesigned and made modified bouganin proteins that have reducedpropensity to activate human T cells compared to the non-modifiedbouganin protein.

(A) Modified Bouganin Proteins

The present invention relates to a modified bouganin protein whereinbouganin has been modified in order to have a reduced propensity toelicit an immune response, preferably a T-cell response, as compared toa non-modified bouganin protein. Mature bouganin protein is a singlepolypeptide of 250 amino acids with a molecular weight of approximately26,200 Da [Den Hartog et al (2002) Eur. J. Biochem. 269: 1772-1779; U.S.Pat. No. 6,680,296]. Bouganin is a type 1 ribosome inactivating protein(RIP) originally isolated from the plant Bougainvillea spectabilis Willd[Bolognesi et al (1997) Planta 203: 422-429]. The RIPs from plants areRNA N-glycosidases that depurinate the major ribosomal RNA of cells,thereby damaging the ribosomes and leading to a cessation of proteinsynthesis and cell death.

The amino acid sequence of the mature bouganin protein (depicted insingle-letter code) is:

[SEQ ID NO. 1] YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKVDRKDLELGVYKLEFSIEAIHGKTINGQEIAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPD MGILKFKSSK.

The term “non-modified bouganin protein” means a bouganin protein thathas not been modified in order to reduce its propensity to elicit animmune response. The sequence of wild-type or a non-modified bouganin isshown in SEQ ID NO:1. However, one of skill in the art will appreciatethat the term “non-modified bouganin” also includes modifications to SEQID NO:1 as long as such modifications do not reduce the propensity toelicit an immune response. Examples of modifications that can be made toSEQ ID NO:1 include peptide fragments and conservative amino acidsubstitutions that do not reduce the immunogenicity of the protein.

The term “modified bouganin protein” means a bouganin protein that hasbeen modified as compared to the non-modified bouganin protein(described above) wherein said modification reduces the propensity ofthe bouganin to elicit an immune response. Modified bouganin protein canalso be referred to as deimmunized bouganin. The “modified bouganinprotein” can be a modified full length sequence or a modified fragmentof the non-modified bouganin protein. The “modified bouganin protein”may also contain other changes as compared to the wild-type bouganinsequence which do not alter immunogenicity of the peptide. The modifiedbouganin protein will preferably have the same biological activity asthe non-modified bouganin.

The term “reduced propensity to elicit an immune response” as usedherein means that the modified bouganin protein is less immunogenic thannon-modified bouganin.

The term “immune response” includes both cellular and humoral immuneresponses. In a preferred embodiment, the modified bouganin has areduced propensity to activate T-cells.

The term “reduced propensity to activate human T-cells” as used hereinmeans the modified bouganin protein has a reduced propensity to activatehuman T-cells as compared to the non-modified bouganin protein. One ofskill in the art can test whether or not a modified bouganin has areduced propensity to activate T-cells using assays known in the artincluding assessing the stimulation index of the protein.

The term “stimulation index” as used herein refers to the measure of theability of the modified or non-modified bouganin protein to activatehuman T cells. For example, the modified or non-modified bouganinprotein, or peptides thereof, can be tested for their ability to evoke aproliferative response in human T-cells cultured in vitro. Where thistype of approach is conducted using naïve human T-cells taken fromhealthy donors, the inventors have established that in the operation ofsuch an assay, a stimulation index equal to or greater than 2.0 is auseful measure of induced proliferation. The stimulation index isconventionally derived by division of the proliferation score (e.g.counts per minute of radioactivity if using ³H-thymidine incorporation)measured to the test peptide by the score measured in cells notcontacted with a test peptide.

In one embodiment, the invention provides a modified bouganin protein,wherein the modified bouganin protein has biological activity and hasreduced propensity to activate human T cells compared to a non-modifiedbouganin protein.

In another embodiment, the invention provides a modified bouganinprotein, wherein the modified bouganin protein has reduced propensity toactivate human T cells compared to a non-modified bouganin protein andhas biological activity that is lower than the non-modified bouganinprotein. In yet another embodiment, the invention provides a modifiedbouganin protein wherein the modified bouganin protein has reducedpropensity to activate human T cells and no biological activity. Suchmodified proteins could, for instance, be used as controls, in assays orto tolerize subjects.

The term “biological activity” as used herein is the ability of themodified or non-modified bouganin protein to inhibit protein synthesison ribosomes, which can be assessed in a number of ways. It should benoted that a modified bouganin protein will still have biologicalactivity even if such activity is lower than that of the non-modifiedprotein, however it would need to have some level of detectableactivity. For example, the biological activity of the modified ornon-modified bouganin protein can be assessed by identifying theirN-glycosidase activity, and in particular with sufficient activity toprovide significant inhibition of protein translation. One such suitableassay involves testing the activity of the variant bouganin proteins incomparison to non-modified bouganin in a cell-free protein synthesisassay. A coupled transcription/translation mix containing methionine,DNA encoding the reporter protein luciferase and serial dilutions ofnon-modified and modified bouganin protein are co-incubated. The levelsof translated luciferase are readily detected using a luminescencecounter following addition of a substrate reagent. The measuredluminescence is inversely proportional to the bouganin N-glycosidaseactivity present in the reaction. It is usual to provide a negativecontrol such as an in-active bouganin protein, for example containing aY70A substitution.

In a preferred embodiment, the modified bouganin peptide is modified atone or more T-cell epitopes in the bouganin protein sequence.

The term “T-cell epitope” means an amino acid sequence which is able tobind major histocompatibility complex (MHC) class II, able to stimulateT-cells and/or also able to bind (without necessarily measurablyactivating) T-cells in complex with MHC class II.

In one aspect, a general method that can be used in the presentinvention leading to the modified bouganin proteins comprising modifiedT-cell epitopes comprises the following steps:

-   -   (i) determining the amino acid sequence of the protein or part        thereof;    -   (ii) identifying one or more potential T-cell epitopes within        the amino acid sequence of the protein by methods such as        determination of the binding of the peptides to MHC molecules        using in vitro or in silico techniques or biological assays;    -   (iii) designing new sequence variants with one or more amino        acids within the identified potential T-cell epitopes modified        in such a way to substantially reduce or eliminate the activity        of the T-cell epitope as determined by the binding of the        peptides to MHC molecules using in vitro or in silico techniques        or biological assays. Such sequence variants are created in such        a way to avoid creation of new potential T-cell epitopes by the        sequence variations unless such new potential T-cell epitopes        are, in turn, modified in such a way to substantially reduce or        eliminate the activity of the T-cell epitope;    -   (iv) constructing such sequence variants by recombinant DNA        techniques and testing said variants in order to identify one or        more variants with desirable properties according to well known        recombinant techniques; and    -   (v) optionally repeating steps (ii) to (iv).

In an example, step (iii) is carried out by substitution, addition ordeletion of amino acid residues in any of the T-cell epitopes in thenon-modified bouganin protein. In another example, the method to makethe modified bouganin protein is made with reference to the homologousprotein sequence and/or in silico modeling.

The identification of potential T-cell epitopes according to step (ii)can be carried out according to methods described previously in the art.Suitable methods are disclosed in WO 98/59244; WO 98/52976; WO 00/34317;WO 02/069232 and may be used to identify binding propensity of bouganinderived peptides to an MHC class II molecule. In order to identifybiologically relevant peptides, the inventors have developed an approachexploiting ex vivo human T-cell proliferation assays. This approach hasproven to be a particularly effective method and has involved thetesting of overlapping bouganin derived peptide sequences in a scheme soas to scan and test the entire bouganin sequence. The synthetic peptidesare tested for their ability to evoke a proliferative response in humanT-cells cultured in vitro. Where this type of approach is conductedusing naïve human T-cells taken from healthy donors, the inventors haveestablished that in the operation of such an assay, a stimulation indexequal to or greater than 2.0 is a useful measure of inducedproliferation. The stimulation index is conventionally derived bydivision of the proliferation score (e.g. counts per minute ofradioactivity if using ³H-thymidine incorporation) measured to the testpeptide by the score measured in cells not contacted with a testpeptide.

Accordingly, in the present studies, 89 synthetic 15-mer peptides (aslisted in Table 1) were used in T-cell proliferation assays with PBMCs(peripheral blood mononuclear cells) from naïve donors (i.e. no knownsensitization to bouganin). 20 donor PBMC samples were selected toachieve an optimal coverage of MHC class II allotypes. PBMCs werestimulated with individual peptides in triplicate cultures for 7 daysbefore proliferation was assessed by ³H-thymidine incorporation. Allpeptides were diluted at two different concentrations: 1 μM and 5 μM.The stimulation indices (SI) were calculated as the amount of ³Hincorporated into the cells, divided by the amount of ³H incorporated inmock-stimulated controls.

This method has identified the most immunogenic regions of the bouganinmolecule in humans. Accordingly, in a specific embodiment, the modifiedbouganin protein is modified at one or more amino acid residues in aT-cell epitope selected from the group consisting of:

(SEQ ID NO: 2) a) AKVDRKDLELGVYKL, termed herein epitope region R1; (SEQID NO: 3) b) LGVYKLEFSIEAIHG, termed herein epitope region R2; and (SEQID NO: 4) c) NGQEIAKFFLIVIQM, termed herein epitope region R3.

These T-cell epitopes have been identified on the basis of giving SI>2in two or more donor PBMC samples. The above disclosed peptide sequencesrepresent the critical information required for the construction ofmodified bouganin proteins in which one or more of these epitopes iscompromised.

In an embodiment of the invention, the modified bouganin protein of theinvention has at least one T-cell epitope removed. In anotherembodiment, the modified bouganin protein of the invention has one, twoor three T-cell epitopes removed. The invention also contemplates amodified bouganin protein wherein 1 to 9 amino acid residues aremodified, preferably in the T-cell epitope. In another embodiment, 1 to5 amino acid residues are modified. The term “modified” as used hereinmeans the amino acid residues are modified by substitution, addition ordeletion, preferably by substitution, but the bouganin protein hasreduced propensity to activate human T cells. In another embodiment themodified protein has biological activity. More preferably the modifiedbouganin protein of the invention is modified by substitution at aposition corresponding to any of the amino acids specified withinsequences (a), (b) or (c) above.

One embodiment of the present invention comprises bouganin proteins forwhich the MHC class II ligands identified within any of the epitopesR1-R3 are modified such as to eliminate binding or otherwise reduce thenumbers of MHC allotypes to which the peptide can bind. Amino acids inthe R1 to R3 regions to eliminate binding or otherwise reduce thenumbers of MHC allotypes to which the peptide can bind can be modifiedby substitution, addition or deletion.

For the elimination of T-cell epitopes, amino acid substitutions aremade at appropriate points within the peptide sequence predicted toachieve substantial reduction or elimination of the activity of theT-cell epitope. In practice an appropriate point will in one embodimentequate to an amino acid residue binding within one of the pocketsprovided within the MHC class II binding groove.

In one embodiment, the binding within the first pocket of the cleft atthe so-called P1 or P1 anchor position of the peptide is modified. Thequality of binding interaction between the P1 anchor residue of thepeptide and the first pocket of the MHC class II binding groove isrecognized as being a major determinant of overall binding affinity forthe whole peptide. An appropriate substitution at this position of thepeptide will be for a residue less readily accommodated within thepocket, for example, substitution to a more hydrophilic residue. Aminoacid residues in the peptide at positions equating to binding withinother pocket regions within the MHC binding cleft are also consideredand fall under the scope of the present.

It is understood that single amino acid substitutions, deletions oradditions within a given potential T-cell epitope are a preferred routeby which the epitope may be eliminated. Combinations of modifications(i.e. substitutions, deletions and additions) within a single epitopemay be contemplated and for example can be particularly appropriatewhere individually defined epitopes are in overlap with each other as isthe present case where epitope regions R1 and R2 overlap by 5 residues.Moreover, either single amino acid modifications within a given epitopeor in combination within a single epitope may be made at positions notequating to the “pocket residues” with respect to the MHC class IIbinding groove, but at any point within the peptide sequence.Modifications may be made with reference to an homologue structure orstructural method produced using in silico techniques known in the artand may be based on known structural features of the molecule accordingto this invention. All such modifications fall within the scope of thepresent invention.

The epitope regions R1-R3 of bouganin were analyzed for indication ofMHC class II ligands encompassed within their respective sequences. Asoftware tool exploiting the schemes outlined in WO 98/59244 and WO02/069232 was used for this analysis. The software simulates the processof antigen presentation at the level of the peptide MHC class II bindinginteraction to provide a binding score for any given peptide sequence.Such a score is determined for many of the predominant MHC class IIallotypes existent in the population. As this scheme is able to test anypeptide sequence, the consequences of amino acid substitutions,additions or deletions with respect to the ability of a peptide tointeract with a MHC class II binding groove can be predicted.Consequently new sequence compositions can be designed which containreduced numbers of peptides able to interact with the MHC class II andthereby function as immunogenic T-cell epitopes.

Under this scheme in one embodiment of the invention substitutionswithin epitope region R1 comprise changes at positions V123, D127 and/orE129. Similarly for epitope region R2, in one embodiment thesubstitution is at position Y133. This residue falls into the region ofoverlap between R1 and R2 but substitution at Y133 is sufficient toeliminate the R2 related MHC class II ligand and is not sufficient ofitself to eliminate R1 related MHC class II ligands. For epitope regionR3, in one embodiment of the invention substitutions are to residuesE151, and/or 1152.

In all instances the substitutions are to one or more alternative aminoacid residues. Analysis of R1 with the MHC II stimulation softwareindicated that amino acid residues 123, 127, 129 and 131 were keyresidues in this epitope for binding to MHC II molecules. Residue 123 isa preferred site for mutation of the R1 region because it is at thesurface of the molecule, away from the active site and is variable inRIP sequence alignment. Nevertheless, not all substitution yield anactive molecule hence the need to validate mutations in the bioactivityassay. Thus for example within R1, substitutions V123T, V123A and V123Qare examples of preferred alternative substitutions. Residue 131 wasfound to be absolutely conserved in RIP and hence is unlikely suitablefor mutation. Residue 127 and 129 are not highly conserved but only arestricted number of residues were found to have an impact on MHC IIbinding. The substitution sets: D127G, D127A, E129Q and E129G are alsopreferred substitutions. For R2, residue 133 was shown to be a likelycandidate to abolish MHC II binding and its apparent surfacelocalization (as determined by modeling) combined to the fact that it isnot highly conserved across RIP make it a good candidate for mutation.Preferred alternative substitutions were found to be Y133N, Y133T,Y133A, Y133R, Y133D, Y133E, Y133Q, Y133G, Y133K, Y133H and Y133S. ForR3, amino acid residues 152, 155 and 158 were identified as key residuesfor MHC II binding. However, residues 155 and 158 are part of a highlyconserved hydrophobic stretch thus suggesting that their mutation wouldnot yield bioactive molecules. Residue poorly conserved was found to bea more likely candidate. For R3, the substitution sets: 1152Q and 1152Aare also preferred substitutions.

Accordingly, the invention provides a modified bouganin protein whereinthe bouganin is modified at one or more of X′, X², X³, X⁴ or X⁵ asfollows:

(epitope region R1, SEQ ID NO: 5) a) AKX¹DRKX²LX³LGVX⁴KL; (epitoperegion R2, SEQ ID NO: 6) b) LGVX⁴KLEFSIEAIHG; and (epitope region R3,SEQ ID NO: 7) c) NGQEX⁵AKFFLIVIQMwherein X¹ through X⁵ can be any amino acid.

In a specific embodiment, X′ is T or A or Q; X² is G or A; X³ is Q or G;X⁴ is N or D or T or A or R or Q or E or G or H or K or S; and X⁵ is Qor A (epitope region R1, SEQ ID NO:8; epitope region R2, SEQ ID NO:9;epitope region R3, SEQ ID NO:10).

Taken together a most preferred substitution set may be compiled basedon immunogenic epitope mapping studies using ex vivo T-cell assays, insilico MHC peptide binding simulations and structural considerationsfrom sequence homology analysis. Finally, if a bioactive protein ispreferred, in vitro activity assay can then be performed on the modifiedprotein that may comprise one or multiple mutations.

Accordingly, in another embodiment, the invention provides a modifiedbouganin peptide, comprising the amino acid sequence:

YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKX ¹DRKX ²LX ³LGVX ⁴KLEFSIEA IHGKTINGQEX⁵AKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQI SPDMGILKFKSSK

wherein X¹ through X⁵ can be any amino acid (SEQ ID NO:11).

In a preferred embodiment, X¹ is T or A or Q; X² is G or A; X³ is Q orG; X⁴ is N or D or T or A or R or Q or E or G or H or K or S; and X⁵ isQ or A (SEQ ID NO: 12).

In a specific embodiment, the modified bouganin protein comprises theamino acid sequence:

(SEQ ID NO: 13) YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAK A DRK A LELGV N KLEFSIEAIH GKTINGQE AAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPD MGILKFKSSK.

In yet another embodiment, the modified bouganin protein comprises theamino acid sequence:

(SEQ ID NO: 14) YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAK A DRK A LELGV Q KLEFSIEAIH GKTINGQE AAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPD MGILKFKSSK.Underlined residues are substituted residues different from thenon-modified bouganin protein.

As will be clear to the person skilled in the art, multiple alternativesets of modifications could be arrived at which achieve the objective ofremoving undesired epitopes. The resulting sequences would howeverremain broadly homologous with the specific proteins disclosed hereinand therefore fall under the scope of the present invention. Obviouschemical equivalents to the sequences disclosed by the present inventionare also contemplated to fall within the scope of the present invention.Such equivalents include proteins that perform substantially the samefunction in substantially the same way.

In another embodiment the modified bouganin protein of the invention has1, 2, 3, 4, 5 or more amino acid modifications in the T-cell epitopes ofthe protein.

In an additional embodiment, the modified bouganin protein of theinvention when tested in a T-cell assay evokes a reduced stimulationindex in comparison to the non-modified bouganin protein.

In a further embodiment of the invention, the T-cell epitopes of thebouganin protein are mapped using a T-cell assay and then modified suchthat upon re-testing in the T-cell assay the modified bouganin proteinevokes a stimulation index less than the non-modified bouganin protein,preferably the stimulation index is less than 2.0.

It will be clear to a person skilled in the art that if the modifiedbouganin protein has substantially reduced or no biological activity, itmay need further modification by substitution, addition or deletion ofamino acid residues to restore the biological activity of the modifiedbouganin protein. However, such modified bouganin proteins that havesubstantially reduced or no biological activity are still encompassedwithin the scope of the invention and have utility as controls inassays, or for tolerization.

In one embodiment, the modified bouganin is mutated at the tyrosineresidue at position 70 to yield an inactive bouganin. In a specificembodiment, the tyrosine at position 70 is replaced with alanine. In apreferred embodiment, the modified bouganin has the sequence:

[SEQ ID NO. 129] YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVAVVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKVDRKDLELGVYKLEFSIEAIHGKTINGQEIAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPD MGILKFKSSK.

Under the scheme of the present invention, the epitopes are compromisedby mutation to result in sequences no longer able to function as T-cellepitopes. It is possible to use recombinant DNA methods to achievedirected mutagenesis of the target sequences and many such techniquesare available and well known in the art. In practice a number ofmodified bouganin proteins will be produced and tested for the desiredimmune and functional characteristic. It is particularly important whenconducting modifications to the protein sequence that the contemplatedchanges do not introduce new immunogenic epitopes. This event is avoidedin practice by re-testing the contemplated sequence for the presence ofepitopes and/or of MHC class II ligands by any suitable means.

The modified bouganin proteins of the invention may also contain or beused to obtain or design “peptide mimetics”. “Peptide mimetics” arestructures which serve as substitutes for peptides in interactionsbetween molecules (See Morgan et al (1989), Ann. Reports Med. Chem.24:243-252 for a review). Peptide mimetics include synthetic structureswhich may or may not contain amino acids and/or peptide bonds but retainthe structural and functional features protein of the invention,including biological activity and a reduced propensity to activate humanT cells. Peptide mimetics also include peptoids, oligopeptoids (Simon etal (1972) Proc. Natl. Acad, Sci USA 89:9367).

Peptide mimetics may be designed based on information obtained bysystematic replacement of L-amino acids by D-amino acids, replacement ofside chains with groups having different electronic properties, and bysystematic replacement of peptide bonds with amide bond replacements.Local conformational constraints can also be introduced to determineconformational requirements for activity of a candidate peptide mimetic.The mimetics may include isosteric amide bonds, or D-amino acids tostabilize or promote reverse turn conformations and to help stabilizethe molecule. Cyclic amino acid analogues may be used to constrain aminoacid residues to particular conformational states. The mimetics can alsoinclude mimics of the secondary structures of the proteins of theinvention. These structures can model the 3-dimensional orientation ofamino acid residues into the known secondary conformations of proteins.Peptoids may also be used which are oligomers of N-substituted aminoacids and can be used as motifs for the generation of chemically diverselibraries of novel molecules.

The molecules of this invention can be prepared in any of several waysbut is most preferably conducted exploiting routine recombinant methods.It is a relatively straightforward procedure to use the proteinsequences and information provided herein to deduce a polynucleotide(DNA) encoding any of the preferred protein sequences. This can beachieved for example using computer software tools such as the DNSstarsoftware suite [DNAstar Inc, Madison, Wis., USA] or similar. Any suchDNA sequence with the capability of encoding the preferred polypeptidesof the present or significant homologues thereof, should be consideredas embodiments of this invention.

As a general scheme, genes encoding any of the preferred modifiedbouganin protein sequences can be made using gene synthesis and clonedinto a suitable expression vector. In turn the expression vector isintroduced into a host cell and cells selected and cultured. Theproteins of the invention are purified from the culture medium andformulated into a preparation for therapeutic administration.Alternatively, a wild-type bouganin gene sequence can be obtained forexample following a cDNA cloning strategy using RNA prepared from theroot tissues of the Bougainvillea spectabilis Willd plant. The wild-typegene can be used as a template for mutagenesis and constructionpreferred variant sequences. In this regard it is particularlyconvenient to use the strategy of “overlap extension PCR” as describedby Higuchi et al [Higuchi et al (1988) Nucleic Acids Res. 16: 7351]although other methodologies and systems could be readily applied.

The biological activity of the proteins of the invention can equally beassessed in many ways. In one embodiment, modified bouganin moleculesare identified with N-glycosidase activity, and in particular withsufficient activity to provide significant inhibition of proteintranslation. One such suitable assay involves testing the activity ofthe modified bouganin proteins in comparison to non-modified bouganin ina cell-free protein synthesis assay. A coupled transcription/translationmix containing methionine, DNA encoding the reporter protein luciferaseand serial dilutions of non-modified and modified bouganin proteins areco-incubated. The levels of translated luciferase are readily detectedusing a luminescence counter following addition of a substrate reagent.The measured luminescence is inversely proportional to the bouganinN-glycosidase activity present in the reaction. It is usual to provide anegative control such as an in-active bouganin protein for examplecontaining a Y70A substitution.

Constitution of the preferred and active bouganin molecules may beachieved by recombinant DNA techniques and this includes bouganinmolecules fused with desired antibody or other targeting moieties.Methods for purifying and manipulating recombinant proteins includingfusion proteins are well known in the art. Necessary techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Weir & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Cabs, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994); “Current Protocols inImmunology” (J. E. Coligan et al., eds., 1991).

The proteins and peptides of the invention can be prepared usingrecombinant DNA methods. The proteins of the invention may also beprepared by chemical synthesis using techniques well known in thechemistry of proteins such as solid phase synthesis (Merrifield, 1964,J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution(Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15I and II, Thieme, Stuttgart).

The present invention also provides a purified and isolated nucleic acidmolecule comprising a sequence encoding the modified bouganin proteinsor peptides of the invention, preferably a sequence encoding the proteindescribed herein as SEQ ID NO:13 or SEQ ID NO:14.

The term “isolated and purified” as used herein refers to a nucleic acidsubstantially free of cellular material or culture medium when producedby recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. An “isolated and purified”nucleic acid is also substantially free of sequences which naturallyflank the nucleic acid (i.e. sequences located at the 5′ and 3′ ends ofthe nucleic acid) from which the nucleic acid is derived.

The term “nucleic acid” as used herein refers to a sequence ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof, which function similarly. The nucleic acidsequences of the present invention may be ribonucleic (RNA) ordeoxyribonucleic acids (DNA) and may contain naturally occurring basesincluding adenine, guanine, cytosine, thymidine and uracil. Thesequences may also contain modified bases such as xanthine,hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl, and other alkyladenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosineand 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thio-alkyl adenines, 8-hydroxyl adenine andother 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiolguanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

In one embodiment, the purified and isolated nucleic acid moleculecomprises a sequence encoding the proteins or peptides, preferably SEQID NO: 13 or SEQ ID NO: 14, of the invention, comprising

-   -   (a) the nucleic acid sequence, wherein T can also be U;    -   (b) nucleic acid sequences complementary to (a);    -   (c) nucleic acid sequences which are homologous to (a) or (b);    -   (d) a fragment of (a) to (c) that is at least 15 bases,        preferably 20 to 30 bases, and which will hybridize to (a)        to (c) under stringent hybridization conditions; or    -   (e) a nucleic acid molecule differing from any of the nucleic        acids of (a) to (c) in codon sequences due to the degeneracy of        the genetic code.

Further, it will be appreciated that the invention includes nucleic acidmolecules comprising nucleic acid sequences having substantial sequencehomology with the nucleic acid sequences encoding the proteins andpeptides of the invention, and fragments thereof. The term “sequenceshaving substantial sequence homology” means those nucleic acid sequenceswhich have slight or inconsequential sequence variations from thesesequences, i.e., the sequences function in substantially the same mannerto produce functionally equivalent proteins. The variations may beattributable to local mutations or structural modifications.

Nucleic acid sequences having substantial homology include nucleic acidsequences having at least 80%, preferably 90% identity with the nucleicacid sequence encoding the proteins and peptides of the invention.Another aspect of the invention provides a nucleic acid molecule, andfragments thereof having at least 15 bases, which hybridize to nucleicacid molecules of the invention under hybridization conditions,preferably stringent hybridization conditions. Appropriate stringencyconditions which promote DNA hybridization are known to those skilled inthe art, or may be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the following maybe employed: 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C. The stringency may be selectedbased on the conditions used in the wash step. For example, the saltconcentration in the wash step can be selected from a high stringency ofabout 0.2×SSC at 50° C. In addition, the temperature in the wash stepcan be at high stringency conditions, at about 65° C.

Accordingly, nucleic acid molecules of the present invention having asequence which encodes a protein or peptide of the invention may beincorporated according to procedures known in the art into anappropriate expression vector which ensures good expression of theprotein or peptide. Possible expression vectors include but are notlimited to cosmids, plasmids, or modified viruses (e.g., replicationdefective retroviruses, adenoviruses and adeno associated viruses), solong as the vector is compatible with the host cell used. The expression“vectors suitable for transformation of a host cell”, means that theexpression vectors contain a nucleic acid molecule of the invention andregulatory sequences, selected on the basis of the host cells to be usedfor expression, which are operatively linked to the nucleic acidmolecule. “Operatively linked” is intended to mean that the nucleic acidis linked to regulatory sequences in a manner which allows expression ofthe nucleic acid.

The invention therefore contemplates a recombinant expression vector ofthe invention containing a nucleic acid molecule of the invention, or afragment thereof, and the necessary regulatory sequences for thetranscription and translation of the inserted protein-sequence. Suitableregulatory sequences may be derived from a variety of sources, includingbacterial, fungal, or viral genes (For example, see the regulatorysequences described in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Selection ofappropriate regulatory sequences is dependent on the host cell chosen,and may be readily accomplished by one of ordinary skill in the art.Examples of such regulatory sequences include: a transcriptionalpromoter and enhancer or RNA polymerase binding sequence, a ribosomalbinding sequence, including a translation initiation signal.Additionally, depending on the host cell chosen and the vector employed,other sequences, such as an origin of replication, additional DNArestriction sites, enhancers, and sequences conferring inducibility oftranscription may be incorporated into the expression vector. It willalso be appreciated that the necessary regulatory sequences may besupplied by the native protein and/or its flanking regions.

The recombinant expression vectors of the invention may also contain aselectable marker gene which facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asG418 and hygromycin which confer resistance to certain drugs,R-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. Transcription of the selectable marker gene is monitored bychanges in the concentration of the selectable marker protein such asβ-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. If the selectable marker gene encodes a protein conferringantibiotic resistance such as neomycin resistance transformant cells canbe selected with G418. Cells that have incorporated the selectablemarker gene will survive, while the other cells die. This makes itpossible to visualize and assay for expression of recombinant expressionvectors of the invention and in particular to determine the effect of amutation on expression and phenotype. It will be appreciated thatselectable markers can be introduced on a separate vector from thenucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion moiety which provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of a target recombinant protein by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beadded to the target recombinant protein to allow separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The term “transformed host cell” isintended to include prokaryotic and eukaryotic cells which have beentransformed or transfected with a recombinant expression vector of theinvention. The terms “transformed with”, “transfected with”,“transformation” and “transfection” are intended to encompassintroduction of nucleic acid (e.g. a vector) into a cell by one of manypossible techniques known in the art. Prokaryotic cells can betransformed with nucleic acid by, for example, electroporation orcalcium-chloride mediated transformation. Nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other such laboratorytextbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the proteins of the invention may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells. Other suitable host cells can be foundin Goeddel, Gene Expression Technology Methods in Enzymology 185,Academic Press, San Diego, Calif. (1991).

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

In some embodiments the expression vector comprises a nucleic acidsequence encoding a modified bouganin with a reduced number of potentialT cell epitopes, operably linked to an expression control sequence. Invarious embodiments the expression vector comprises a nucleic acidsequence encoding the proteins or peptides of the invention, or adegenerate variant thereof and will comprise at least the RIP encodingdomain of the said nucleic acids operably linked with suitableexpression control and selection sequences. Degeneracy in relation topolynucleotides refers to the fact well recognized that in the geneticcode many amino acids are specified by more than one codon. Thedegeneracy of the code accounts for 20 different amino acids encoded by64 possible triplet sequences of the four different bases comprisingDNA.

The term “RIP encoding domain” or “Ribosome Inactivating Proteinencoding domain” as used here in means the functional domain which givesbouganin its biological activity.

The nucleic acid molecules of the invention may also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing polydeoxynucleotides are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

The invention also provides nucleic acids encoding fusion proteinscomprising a novel protein of the invention and a selected protein, or aselectable marker protein

Another aspect of the present invention is a cultured cell comprising atleast one of the above-mentioned vectors.

A further aspect of the present invention is a method for preparing themodified bouganin comprising culturing the above mentioned cell underconditions permitting expression of the modified bouganin from theexpression vector and purifying the bouganin from the cell.

(B) Modified Bouganin Cytotoxins:

As mentioned previously, bouganin is a type 1 ribosome inactivatingprotein (RIP) that depurinates the major ribosomal RNA of cells leadingto cessation of protein synthesis and cell death. As such, the modifiedbouganins of the invention can be used to prepare cytotoxins. Cytotoxinscontaining a modified bouganin protein are preferred over cytotoxinscontaining a non-modified bouganin protein as the former is lessimmunogenic and will be less likely to be destroyed by the immune systembefore it reaches its target.

Accordingly, the present invention also provides a cytotoxin comprising(a) a targeting moiety attached to (b) a modified bouganin protein ofthe invention.

The term “modified bouganin protein of the invention” is used for easeof referral and includes any and all of the modified bouganin proteinsdescribed herein such as the modified bouganin proteins described abovein Section (A) as well as in the figures and examples.

The term “targeting moiety” as used herein refers to a substance, means,or technique of delivering the modified bouganin protein to a targetcell. In one embodiment the targeting moiety is an antibody. In oneembodiment the targeting moiety could be a liposome. In one embodimentthe liposome can be linked to an antibody. In another embodiment thetargeting moiety is a protein able to direct a specific bindinginteraction to a particular target cell. Such protein moieties include avariety of polypeptide ligands for which there are specific cell surfacereceptors and include therefore numerous cytokines, peptide andpolypeptide hormones and other biological response modifiers. Prominentexamples include such proteins as vascular epithelial growth factor,epidermal growth factor, heregulin, the interleukins, interferons,tumour necrosis factor and other protein and glycoprotein molecules.Fusion proteins of these and other molecules with bouganin of thepresent invention may be contemplated and may comprise the modifiedbouganin moiety in either the N-terminal or C-terminal orientation withrespect to the protein ligand domain. The targeting moiety may bejointed directly to the proteins of the invention or through a linker.In one embodiment, the linker is a peptide linker or a chemical linker.Equally, chemical cross-linking of the purified ligand to the modifiedbouganin protein may be contemplated and within the scope of the presentinvention.

In a preferred embodiment, the present invention provides a cytotoxincomprising (a) a ligand that binds to a cancer cell attached to; (b) amodified bouganin protein of the invention.

The ligand can be any molecule that can bind to a cancer cell including,but not limited to, proteins. In one embodiment, the ligand is anantibody or antibody fragment that recognizes the surface of a cancercell.

Accordingly, the cytotoxins of the present invention may be used totreat various forms of cancer such as colorectal cancer, breast cancer,ovarian cancer, pancreatic cancer, head and neck cancer, bladder cancer,gastrointestinal cancer, prostate cancer, small cell and non small celllung cancer, sarcomas, gliomas, T- and B-cell lymphomas.

In one embodiment, the cancer cell binding ligand comprises a completeimmunoglobulin molecule that binds to the cancer cell. When a cancercell binding ligand is an antibody or fragment thereof, cytotoxin can bereferred to as immunotoxin. In another embodiment, the cancercell-binding ligand is a dimer of Fab, Fab′, scFv, single-domainantibody fragments, or disulfide stabilized Fv fragments. In anotherembodiment, the cancer antibody comprises a variable heavy chain,variable light chain, Fab, Fab′, scFv, single-domain antibody fragment,or disulfide-stabilized Fv fragment. Portions of the cancer cell-bindingligand may be derived from one or more species, preferably comprisingportions derived from the human species, and most preferably arecompletely human or humanized. Regions designed to facilitatepurification or for conjugation to toxin may also be included in oradded to the cancer cell-binding portion.

In a particular embodiment, the cancer cell binding ligand recognizesEp-CAM. Ep-CAM (for Epithelial Cell Adhesion Molecule, which is alsoknown as 17-1A, KSA, EGP-2 and GA733-2) is a transmembrane protein thatis highly expressed in many solid tumors, including carcinomas of thelung, breast, ovary, colorectum, and squamous cell carcinoma of the headand neck, but weakly expressed in most normal epithelial tissues.

Accordingly, in one embodiment, the invention provides anEp-CAM-targeted-modified bouganin cytotoxin comprising (a) a ligand(such as an antibody or antibody fragment) that binds to Ep-CAM on thecancer cell attached to; (b) a modified bouganin protein having areduced propensity to activate T-cells as compared to a non-modifiedbouganin protein.

In a specific embodiment, the cytotoxin comprises (a) a humanizedantibody or antibody fragment that binds to the extracellular domain ofhuman Ep-CAM and comprises complementarity determining region (CDR)sequences derived from a MOC-31 antibody attached to: (b) a modifiedbouganin protein having a reduced propensity to activate T-cells ascompared to a non-modified bouganin protein.

Suitable Ep-CAM-targeted-modified bouganins according to the inventioninclude, without limitation, VB6-845 and variants thereof, othercytotoxins that comprises other single or double chain immunoglobulinsthat selectively bind Ep-CAM, or variants thereof. The term “VB6-845” asused herein means a cytotoxin that comprises a Fab version of ananti-Ep-CAM scFv antibody linked to a modified form of bouganin, Bou 156(SEQ ID NO:13). The amino acid sequence and nucleotide sequence ofVB6-845 is shown in FIG. 3B (SEQ ID NO:16 and SEQ ID NO:15,respectively).

In another embodiment, the cancer cell binding ligand recognizes atumor-associated antigen that is found specifically on neoplastic cellsand not on normal cells. In a preferred embodiment, the ligand is anantibody that binds tumor-associated antigen. Theanti-tumor-associated-antigen antibody specifically recognizes cancercells from a wide variety of cancers but does not recognize normal,non-cancerous cells.

Accordingly in another embodiment, the invention provides a cytotoxincomprising (a) ligand (such as an antibody or antibody fragment) thatbinds to tumor-associated antigen on the cancer cell attached to; (b) amodified bouganin protein having a reduced propensity to activateT-cells as compared to a non-modified bouganin protein.

Suitable tumor-associated-antigen-targeted-modified bouganins accordingto the invention include, without limitation, VB6-011 and variantsthereof, other cytotoxins that comprises other single or double chainimmunoglobulins that selectively bind tumor-associated-antigen, orvariants thereof. The term “VB6-011” as used herein means a cytotoxinthat comprises a Fab version of the H11 human monoclonal antibodygenetically linked to a modified form of bouganin, BOU 156 (SEQ ID No.13). The H11 antibody was obtained by the fusion of peripheral bloodlymphocytes of a 64 year old male cancer patient fused with a humanmyeloma cell line to produce hybridomas. The hybridoma NBGM1/H11produces an IgM_(k) that was re-engineered into a Fab format to makeVB6-011 (see U.S. Pat. No. 6,207,153 or WO 97/44461 for detail on thepreparation of the H11 antibody-secreting hybridoma). The amino acidsequence and nucleotide sequence of VB6-011 is shown in FIG. 15 (SEQ IDNO:28 and SEQ ID NO:27, respectively).

In a specific, non-limiting embodiment, the cytotoxin comprises VB6-845(FIG. 3B, SEQ ID No.16) or VB6-011 (FIG. 15, SEQ ID NO: 28). In othernon-limiting embodiments, the cytotoxin comprises a variant of VB6-845or VB6-011.

A VB6-845 variant binds to the same Ep-CAM epitope or to a substantiallysimilar Ep-CAM epitope that is bound by VB6-845, and the variant maycompetitively inhibit VB6-845 binding to Ep-CAM, under physiologicconditions, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. A VB6-845 variant maycomprise the same modified bouganin as VB6-845, or may comprise adifferent modified bouganin of the invention. In another non-limitingembodiment, the cytotoxin comprises an Ep-CAM-binding portion comprisingthe variable region of MOC31, or a variant thereof. In yet anotherembodiment, the cytotoxin comprises an Ep-CAM-binding portion comprising4D5MOCB, or a variant thereof. Binding of any of these cytotoxins toEp-CAM may be reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% by competitionwith the reference MOC31 or 4D5MOCB antibody under physiologicconditions.

A VB6-011 variant binds to the same tumor-associated-antigen epitope orto a substantially similar tumor-associated-antigen epitope that isbound by VB6-011, and the variant may competitively inhibit VB6-011binding to tumor-associated-antigen, under physiologic conditions, by atleast 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95%. A VB6-011 variant may comprise the samemodified bouganin as VB6-011, or may comprise a different modifiedbouganin of the invention.

In another non-limiting embodiment, the cytotoxin comprises atumor-associated-antigen binding portion comprising the H11 monoclonalantibody, H11 antigen binding fragments, or variants thereof. Binding ofany of these cytotoxins to VB6-011 may be reduced by at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95% by competition with the reference H11 antibody underphysiologic conditions.

In a preferred embodiment, the binding affinity of the Ep-CAM-bindingportion or the tumor-associated-antigen-binding portion is at least fourorders of magnitude, preferably at least three orders of magnitude, morepreferably less than two orders of magnitude of the binding affinity ofVB6-845 or VB6-011 respectively as measured by standard laboratorytechniques. In non-limiting embodiments, the Ep-CAM-binding portion maycompetitively block the binding of a known anti-Ep-CAM antibody, suchas, but not limited to, PANOREX® or MT201, to Ep-CAM, under physiologicconditions, by at least 0.1%, 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Innon-limiting embodiments, the tumor-associated-antigen-binding portionmay competitively block the binding of a knownanti-tumor-associated-antigen antibody, such as, but not limited to,H11, to tumor-associated antigen, under physiologic conditions, by atleast 0.1%, 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95%.

The skilled artisan would appreciate that specificity determiningresidues can be identified. The term “specificity determining residue,”also known as “SDR,” refers to a residue that forms part of the paratopeof an antibody, particularly CDR residues, the individual substitutionof which by alanine, independently of any other mutations, diminishesthe affinity of the antibody for the epitope by at least 10 fold,preferably by at least 100 fold, more preferably by at least 1000 fold.This loss in affinity underscores that residue's importance in theability of the antibody to bind the epitope. See, e.g., Tamura et al.,2000, “Structural correlates of an anticarcinoma antibody:identification of specificity-determining residues (SDRs) anddevelopment of a minimally immunogenic antibody variant by retention ofSDRs only,” J. Immunol. 164(3):1432-1441.

The effect of single or multiple mutations on binding activity,particularly on binding affinity, may be evaluated contemporaneously toassess the importance of a particular series of amino acids on thebinding interaction (e.g., the contribution of the light or heavy chainCDR2 to binding). Effects of an amino acid mutation may also beevaluated sequentially to assess the contribution of a single amino acidwhen assessed individually. Such evaluations can be performed, forexample, by in vitro saturation scanning (see, e.g., U.S. Pat. No.6,180,341; Hilton et al., 1996, “Saturation mutagenesis of the WSXWSmotif of the erythropoietin receptor,” J Biol. Chem. 271:4699-4708) andsite-directed mutagenesis (see, e.g., Cunningham and Wells, 1989,“High-resolution epitope mapping of hGH-receptor interactions byalanine-scanning mutagenesis,” Science 244:1081-1085; Bass et al., 1991,“A systematic mutational analysis of hormone-binding determinants in thehuman growth hormone receptor,” Proc Natl Acad Sci. USA 88:4498-4502).In the alanine-scanning mutagenesis technique, single alanine mutationsare introduced at multiple residues in the molecule, and the resultantmutant molecules are tested for biological activity to identify aminoacid residues that are critical to the activity of the molecule.

Sites of ligand-receptor or other biological interaction can also beidentified by physical analysis of structure as determined by, forexample, nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids (see, e.g., de Vos et al., 1992,“Human growth hormone and extracellular domain of its receptor: crystalstructure of the complex,” Science 255:306-312; Smith et al., 1992,“Human interleukin 4. The solution structure of a four-helix bundleprotein,” J Mol. Biol. 224:899-904; Wlodaver et al., 1992, “Crystalstructure of human recombinant interleukin-4 at 2.25 A resolution,” FEBSLett. 309:59-64). Additionally, the importance of particular individualamino acids, or series of amino acids, may be evaluated by comparisonwith the amino acid sequence of related polypeptides or analogousbinding sites.

Furthermore, the skilled artisan would appreciate that increased aviditymay compensate for lower binding affinity. The avidity of a cytotoxinfor a cancer cell receptor is a measure of the strength of theEp-CAM-binding portion's binding of Ep-CAM, which has multiple bindingsites. The functional binding strength between Ep-CAM and theEp-CAM-binding portion represents the sum strength of all the affinitybonds, and thus an individual component may bind with relatively lowaffinity, but a multimer of such components may demonstrate potentbiological effect. In fact, the multiple interactions betweenEp-CAM-binding sites and Ep-CAM epitopes, may demonstrate much greaterthan additive biological effect, i.e., the advantage of multivalence canbe many orders of magnitude with respect to the equilibrium constant.

Similarly, the avidity of a cytotoxin for a cancer cell receptor is ameasure of the strength of the tumor-associated antigen-bindingportion's binding of tumor-associated antigen, which may have multiplebinding sites. The functional binding strength between tumor-associatedantigen and the tumor-associated antigen-binding portion represents thesum strength of all the affinity bonds, and thus an individual componentmay bind with relatively low affinity, but a multimer of such componentsmay demonstrate potent biological effect. In fact, the multipleinteractions between tumor-associated antigen-binding sites andtumor-associated antigen epitopes may demonstrate much greater thanadditive biological effect, i.e., the advantage of multivalence can bemany orders of magnitude with respect to the equilibrium constant.

In one non-limiting embodiment, the Ep-CAM-binding portion has astructure substantially similar to that of 4D5MOCB. The substantiallysimilar structure can be characterized by reference to epitope maps thatreflect the binding points of the cytotoxin's Ep-CAM-binding portion toan Ep-CAM molecule. In another non-limiting embodiment, epitope maps canbe generated for the tumor-associated antigen binding portion and asubstantially similar structure can be characterized by reference toepitope maps that reflect the binding points of the cytotoxin'stumor-associated antigen binding portion to a tumor-associated antigenmolecule.

The cytotoxins of the present invention may be prepared by chemicalsynthesis using techniques well known in the chemistry of proteins suchas solid phase synthesis (Merrifield, J. Am. Chem. Assoc. 85:2149-2154(1964)) or synthesis in homogenous solution (Houbenweyl, Methods ofOrganic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart(1987)). In one embodiment, the cancer-binding ligand and modifiedbouganin are both proteins and can be conjugated using techniques wellknown in the art. There are several hundred crosslinkers available thatcan conjugate two proteins. (See for example “Chemistry of ProteinConjugation and Crosslinking”. 1991, Shans Wong, CRC Press, Ann Arbor).The crosslinker is generally chosen based on the reactive functionalgroups available or inserted on the ligand or toxin. In addition, ifthere are no reactive groups a photoactivatible crosslinker can be used.In certain instances, it may be desirable to include a spacer betweenthe ligand and the toxin. Crosslinking agents known to the art includethe homobifunctional agents: glutaraldehyde, dimethyladipimidate andBis(diazobenzidine) and the heterobifunctional agents: mMaleimidobenzoyl-N-Hydroxysuccinimide and Sulfo-mMaleimidobenzoyl-N-Hydroxysuccinimide.

A ligand-bouganin toxin fusion protein may also be prepared usingrecombinant DNA techniques. In such a case a DNA sequence encoding thecancer-binding ligand is fused to a DNA sequence encoding the modifiedbouganin protein, resulting in a chimeric DNA molecule. The chimeric DNAsequence is transfected into a host cell that expresses theligand-bouganin fusion protein. The fusion protein can be recovered fromthe cell culture and purified using techniques known in the art.

Antibodies having specificity for cell surface proteins such as Ep-CAMand tumor-associated antigen may be prepared by conventional methods. Amammal, (e.g. a mouse, hamster, or rabbit) can be immunized with animmunogenic form of the peptide which elicits an antibody response inthe mammal. Techniques for conferring immunogenicity on a peptideinclude conjugation to carriers or other techniques well known in theart. For example, the peptide can be administered in the presence ofadjuvant. The progress of immunization can be monitored by detection ofantibody titers in plasma or serum. Standard ELISA or other immunoassayprocedures can be used with the immunogen as antigen to assess thelevels of antibodies. Following immunization, antisera can be obtainedand, if desired, polyclonal antibodies isolated from the sera.

To produce monoclonal antibodies, antibody-producing cells (lymphocytes)can be harvested from an immunized animal and fused with myeloma cellsby standard somatic cell fusion procedures thus immortalizing thesecells and yielding hybridoma cells. Such techniques are well known inthe art, (e.g. the hybridoma technique originally developed by Kohlerand Milstein (Nature 256:495-497 (1975)) as well as other techniquessuch as the human B-cell hybridoma technique (Kozbor et al., Immunol.Today 4:72 (1983)), the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies in CancerTherapy Allen R., Bliss, Inc., pages 77-96 (1985)), and screening ofcombinatorial antibody libraries (Huse et al., Science 246:1275 (1989)).Hybridoma cells can be screened immunochemically for production ofantibodies specifically reactive with the peptide and the monoclonalantibodies can be isolated.

The term “antibody” as used herein is intended to include monoclonalantibodies and polyclonal antibodies, antibody fragments (e.g. Fab andF(ab′)₂, and single chain antibodies (scFv)), and chimeric antibodieswhich also specifically react with a cell surface component. Antibodiescan be fragmented using conventional techniques and the fragmentsscreened for utility in the same manner as described above. For example,F(ab′)₂ fragments can be generated by treating antibody with pepsin. Theresulting F(ab′)₂ fragment can be treated to reduce disulfide bridges toproduce Fab′ fragments. Single chain antibodies combine theantigen-binding regions of an antibody on a single stably foldedpolypeptide chain. Single chain antibodies can be generated byrecombinant technology.

Chimeric antibody derivatives, i.e., antibody molecules that combine anon-human animal variable region and a human constant region are alsocontemplated within the scope of the invention. Chimeric antibodymolecules can include, for example, the antigen binding domain from anantibody of a mouse, rat, or other species, with human constant regions.Conventional methods may be used to make chimeric antibodies containingthe immunoglobulin variable region which recognizes a cell surfaceantigen (See, for example, Morrison et al., Proc. Natl. Acad. Sci.U.S.A. 81:6851 (1985); Takeda et al., Nature 314:452 (1985), Cabilly etal., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397;Tanaguchi et al., E. P. Patent No. 171,496; European Patent No. 173,494,United Kingdom Patent No. GB 2177096B). It is expected that chimericantibodies would be less immunogenic in a human subject than thecorresponding non-chimeric antibody. Chimeric antibodies can bestabilized by the method described in Pluckthun et al., WO 00/61635.

Monoclonal or chimeric antibodies specifically reactive against cellsurface components can be further humanized by producing human constantregion chimeras, in which parts of the variable regions, particularlythe conserved framework regions of the antigen-binding domain, are ofhuman origin and only the hypervariable regions are of non-human origin.Such immunoglobulin molecules may be made by techniques known in theart, (e.g. Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80:7308-7312(1983); Kozbor et al., Immunology Today 4:7279 (1983); Olsson et al.,Meth. Enzymol., 92:3-16 (1982), and PCT Publication WO92/06193 or EP239,400). Humanized antibodies can also be commercially produced(Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)In addition, monoclonal or chimeric antibodies specifically reactiveagainst cell surface components can be made less immunogenic by reducingtheir number of potential T-cell epitopes.

Specific antibodies, or antibody fragments, reactive against cellsurface components may also be generated by screening expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with cell surface components. For example, complete Fabfragments, VH regions and Fv regions can be expressed in bacteria usingphage expression libraries (See for example Ward et al., Nature341:544-546 (1989); Huse et al., Science 246:1275-1281 (1989); andMcCafferty et al., Nature 348:552-554 (1990)). Alternatively, a SCID-humouse, for example the model developed by Genpharm, can be used toproduce antibodies, or fragments thereof.

In all instances where a modified bouganin protein is made in fusionwith an antibody sequence it is most desired to use antibody sequencesin which T cell epitopes or sequences able to bind MHC class IImolecules or stimulate T cells or bind to T cells in association withMHC class II molecules have been removed.

A further embodiment of the present invention, the modified bouganinprotein may be linked to a non-antibody protein yet a protein able todirect a specific binding interaction to a particular target cell. Suchprotein moieties include a variety of polypeptide ligands for whichthere are specific cell surface receptors and include therefore numerouscytokines, peptide and polypeptide hormones and other biologicalresponse modifiers. Prominent examples include such proteins as vascularepithelial growth factor, epidermal growth factor, heregulin, theinterleukins, interferons, tumour necrosis factor and other protein andglycoprotein molecules. Fusion proteins of these and other moleculeswith bouganin of the present invention may be contemplated and maycomprise the modified bouganin moiety in either the N-terminal orC-terminal orientation with respect to the protein ligand domain.Equally, chemical cross-linking of the purified ligand to the modifiedbouganin protein may be contemplated and within the scope of the presentinvention.

In a further embodiment the modified bouganin protein of the presentinvention may be used as a complex containing a water soluble polymersuch as hydroxypropylmethacrylamide or other polymers where the modifiedbouganin protein is in covalent attachment to the polymer or in anon-covalent binding interaction with the polymer. Such an embodimentmay additionally include an antigen binding domain such as an antibodyor a fragment of an antibody in combination with the polymer bouganincomplex.

(C) Uses of the Cytotoxins

The modified bouganin proteins of the invention may be used tospecifically inhibit or destroy mammalian cells affected by cancer. Itis an advantage of the cytotoxins of the invention that they have lessimmunogenicity, allowing the RIP to enter the cell and effectively killthe cancer cell. Thus, the cytotoxin may be used to specifically targetcancer cells. The bouganin, once in the cancer cell, depurinates themajor ribosomal RNA, thereby damaging the ribosomes and leading to acessation of protein synthesis and cell death.

Accordingly, in one embodiment, the invention provides a method ofinhibiting or destroying a cancer cell comprising administering acytotoxin of the invention to an animal in need thereof. The presentinvention also includes a use of a cytotoxin of the invention to inhibitor destroy a cancer cell. The present invention further includes a useof a cytotoxin of the invention in the manufacture of a medicament toinhibit or destroy a cancer cell. The type of cancer cells that areinhibited or destroyed by a cytotoxin will be determined by the antigenspecificity of its antibody portion.

In another embodiment, the invention provides a method of inhibiting ordestroying cancer cells comprising the steps of preparing a cytotoxin ofthe invention and administering the cytotoxin to the cells. The cancercan be any type of cancer, including, but not limited to, colorectalcancer, breast cancer, ovarian cancer, pancreatic cancer, head and neckcancer, bladder cancer, liver cancer, renal cancer, melanomas,gastrointestinal cancer, prostate cancer, small cell and non small celllung cancer, sarcomas, gliomas, T- and B-cell lymphomas.

The ability of the cytotoxins of the invention to selectively inhibit ordestroy animal cancer cells may be readily tested in vitro using animalcancer cell lines. The selective inhibitory effect of the cytotoxins ofthe invention may be determined, for example, by demonstrating theselective inhibition of cellular proliferation in cancer cells.

Toxicity may be measured based on cell viability, for example theviability of normal and cancerous cell cultures exposed to thecytotoxins may be compared. Cell viability may be assessed by knowntechniques, such as trypan blue exclusion assays.

In another example, a number of models may be used to test thecytotoxicity of cytotoxins. Thompson, E. W. et al. (Breast Cancer Res.Treatment 31:357-370 (1994)) has described a model for the determinationof invasiveness of human breast cancer cells in vitro by measuringtumour cell-mediated proteolysis of extracellular matrix and tumour cellinvasion of reconstituted basement membrane (collagen, laminin,fibronectin, Matrigel or gelatin). Other applicable cancer cell modelsinclude cultured ovarian adenocarcinoma cells (Young, T. N. et al.Gynecol. Oncol. 62:89-99 (1996); Moore, D. H. et al. Gynecol. Oncol.65:78-82 (1997)), human follicular thyroid cancer cells (Demeure, M. J.et al., World J. Surg. 16:770-776 (1992)), human melanoma (A-2058) andfibrosarcoma (HT-1080) cell lines (Mackay, A. R. et al. Lab. Invest.70:781-783 (1994)), and lung squamous (HS-24) and adenocarcinoma (SB-3)cell lines (Spiess, E. et al. J. Histochem. Cytochem. 42:917-929(1994)). An in vivo test system involving the implantation of tumoursand measurement of tumour growth and metastasis in athymic nude mice hasalso been described (Thompson, E. W. et al., Breast Cancer Res.Treatment 31:357-370 (1994); Shi, Y. E. et al., Cancer Res. 53:1409-1415(1993)).

The present invention also relates to a method of treating cancercomprising administering an effective amount of one or more cytotoxinsof the present invention to an animal in need thereof. The inventionincludes a use of a cytotoxin of the invention to treat cancer. Theinvention further includes a use of a cytotoxin of the invention in themanufacture of a medicament for treating cancer.

The term “animal” includes all members of the animal kingdom, includinghumans.

The term “treating cancer” or “treat cancer” refers to inhibition ofcancer cell replication, inhibition of cancer spread (metastasis),inhibition of tumor growth, reduction of cancer cell number or tumorgrowth, decrease in the malignant grade of a cancer or improvement ofcancer related symptoms.

In a preferred embodiment, the animal is human. In another embodiment,the cancer is selected from the group consisting of colorectal cancer,breast cancer, ovarian cancer, pancreatic cancer, head and neck cancer,bladder cancer, liver cancer, renal cancer, melanomas, gastrointestinalcancer, prostate cancer, small cell and non small cell lung cancer,sarcomas, gliomas and T- and B-cell lymphomas.

Clinical outcomes of cancer treatments using a cytotoxin of theinvention are readily discernible by one of skill in the relevant art,such as a physician. For example, standard medical tests to measureclinical markers of cancer may be strong indicators of the treatment'sefficacy. Such tests may include, without limitation, physicalexamination, performance scales, disease markers, 12-lead ECG, tumormeasurements, tissue biopsy, cytoscopy, cytology, longest diameter oftumor calculations, radiography, digital imaging of the tumor, vitalsigns, weight, recordation of adverse events, assessment of infectiousepisodes, assessment of concomitant medications, pain assessment, bloodor serum chemistry, urinalysis, CT scan, and pharmacokinetic analysis.Furthermore, synergistic effects of a combination therapy comprising thecytotoxin and another cancer therapeutic may be determined bycomparative studies with patients undergoing monotherapy.

Remission malignant tumors may be evaluated using criteria accepted bythe skilled artisan. See, e.g., Therasse et al., 2000, “New guidelinesto evaluate the response to treatment in solid tumors. EuropeanOrganization for Research and Treatment of Cancer, National CancerInstitute of the United States, National Cancer Institute of Canada,” JNatl Cancer Inst. February 2; 92(3):205-16.

The effective dose of a specific cytotoxin construct may depend onvarious factors, including the type of cancer, the size of the tumour,the stage of the cancer, the cytotoxin's toxicity to the patient, thespecificity of targeting to cancer cells, as well as the age, weight,and health of the patient.

Cytotoxins comprising the modified bouganin can be administered by i.v.infusion over a period of minutes to hours, depending on the dose andthe concentration of the cytotoxin in the infusate.

In one embodiment, the cytotoxin is infused over a period of 3 hours.

In one embodiment, the effective dose by i.v. administration ofcytotoxin may range from about 1 to 100 mg/kg/dose. In otherembodiments, the dose may range from approximately 2 to 50 mg/kg/dose.In specific embodiments, the dose may be at least approximately 2, 4, 8,13, 20, 28, 40, 50 mg/kg/dose.

In one embodiment, the single dose is administered approximately everyweek for approximately 1, 2, 3, 4, 5, or 6 weeks. The single dose can beadministered in consecutive weeks or, alternatively, one or more weekscan be skipped. After this cycle, a subsequent cycle may beginapproximately 1, 2, 4, 6, or 12 weeks later. The treatment regime mayinclude 1, 2, 3, 4, 5, 6 or more cycles, each cycle being spaced apartby approximately 1, 2, 4, 6, or 12 weeks.

In another embodiment the single dose is administered every month forapproximately 1, 2, 3, 4, 5, or 6 consecutive months. After this cycle,a subsequent cycle may begin approximately 1, 2, 4, 6, or 12 monthslater. The treatment regime may include 1, 2, 3, 4, 5, 6 or more cycles,each cycle being spaced apart by approximately 1, 2, 4, 6, or 12 months.

In a particular non-limiting embodiment, the effective dose of thecytotoxin is between about 1 and 50 mg/kg/tumor/day, wherein the patientis administered a single dose per day. The single dose is administeredapproximately every day (one or more days may optionally be skipped) forapproximately 1, 2, 3, 4, 5, 6 or 7 consecutive days. After this cycle,a subsequent cycle may begin approximately 1, 2, 3, 4, 5, or 6 weekslater. The treatment regime may include 1, 2, 3, 4, 5, 6 or more cycles,each cycle being spaced apart by approximately 1, 2, 3, 4, 5, or 6weeks. The injection volume preferably is at least an effective amount,which is appropriate to the type and/or location of the tumor. Themaximum injection volume in a single dose may be between about 25% and75% of tumor volume, for example approximately one-quarter, one-third,or three-quarters of the estimated target tumor volume. In a specific,non-limiting embodiment, the maximum injection volume in a single doseis approximately 30% of the tumor volume.

In another embodiment, the cytotoxin is infused for 3 hours at a rate of100 cc per hour with a solution containing from 1 to 10 mg cytotoxin/mL.The cytotoxin will be diluted in a suitable physiologically compatiblesolution.

The effective dose of another cancer therapeutic to be administeredtogether with a cytotoxin during a cycle also varies according to themode of administration. The one or more cancer therapeutics may bedelivered intratumorally, or by other modes of administration.Typically, chemotherapeutic agents are administered systemically.Standard dosage and treatment regimens are known in the art (see, e.g.,the latest editions of the Merck Index and the Physician's DeskReference; NCCN Practice Guidelines in Oncology)).

Combination therapy with a cytotoxin may sensitize the cancer or tumorto administration of an additional cancer therapeutic. Accordingly, thepresent invention contemplates combination therapies for preventing,treating, and/or preventing recurrence of cancer comprisingadministering an effective amount of a cytotoxin prior to, subsequently,or concurrently with a reduced dose of a cancer therapeutic. Forexample, initial treatment with a cytotoxin may increase the sensitivityof a cancer or tumor to subsequent challenge with a dose of cancertherapeutic. This dose is near, or below, the low range of standarddosages when the cancer therapeutic is administered alone, or in theabsence of a cytotoxin. When concurrently administered, the cytotoxinmay be administered separately from the cancer therapeutic, andoptionally, via a different mode of administration.

In another embodiment, a cytotoxin is administered in combination withat least one other immunotherapeutic.

In another embodiment, a cytotoxin is administered in combination with aregimen of radiation therapy. The therapy may also comprise surgeryand/or chemotherapy. For example, the cytotoxin may be administered incombination with radiation therapy and cisplatin (Platinol),fluorouracil (5-FU, Adrucil), carboplatin (Paraplatin), and/orpaclitaxel (Taxol). Treatment with the cytotoxin may allow use of lowerdoses of radiation and/or less frequent radiation treatments, which mayfor example, reduce the incidence of severe sore throat that impedesswallowing function potentially resulting in undesired weight loss ordehydration.

In another embodiment, a cytotoxin is administered in combination withone or more cytokines which include, without limitation, a lymphokine,tumor necrosis factors, tumor necrosis factor-like cytokine,lymphotoxin, interferon, macrophage inflammatory protein, granulocytemonocyte colony stimulating factor, interleukin (including, withoutlimitation, interleukin-1, interleukin-2, interleukin-6, interleukin-12,interleukin-15, interleukin-18), and a variant thereof, including apharmaceutically acceptable salt thereof.

In yet another embodiment, a cytotoxin is administered in combinationwith a cancer vaccine including, without limitation, autologous cells ortissues, non-autologous cells or tissues, carcinoembryonic antigen,alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine,melanocyte lineage proteins, and mutated, tumor-specific antigens.

In yet another embodiment, a cytotoxin is administered in associationwith hormonal therapy. Hormonal therapeutics include, withoutlimitation, a hormonal agonist, hormonal antagonist (e.g., flutamide,tamoxifen, leuprolide acetate (LUPRON)), and steroid (e.g.,dexamethasone, retinoid, betamethasone, cortisol, cortisone, prednisone,dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen,testosterone, progestin).

In yet another embodiment, a cytotoxin is administered in associationwith a gene therapy program to treat or prevent cancer.

In yet another embodiment, an Ep-CAM-targeted cytotoxin is administeredin combination with one or more agents that increase expression ofEp-CAM in the tumor cells of interest. Ep-CAM expression preferably isincreased so that a greater number of Ep-CAM molecules are expressed onthe tumor cell surface. For example, the agent may inhibit the normalcycles of Ep-CAM antigen endocytosis. Such combination treatment mayimprove the clinical efficacy of the Ep-CAM-targeted cytotoxin alone, orwith other cancer therapeutics or radiation therapy. In specific,nonlimiting embodiments, the agent which increases Ep-CAM expression inthe tumor cells is vinorelbine tartrate (Navelbine) and/or paclitax(Taxol). See, e.g., Thurmond et al., 2003, “Adenocarcinoma cells exposedin vitro to Navelbine or Taxol increase Ep-CAM expression through anovel mechanism.” Cancer Immunol Immunother. July; 52(7):429-37.

Combination therapy may thus increase the sensitivity of the cancer ortumor to the administered cytotoxin and/or additional cancertherapeutic. In this manner, shorter treatment cycles may be possiblethereby reducing toxic events. Accordingly, the invention provides amethod for treating or preventing cancer comprising administering to apatient in need thereof an effective amount of a cytotoxin and at leastone other cancer therapeutic for a short treatment cycle. The cycleduration may vary according to the specific cancer therapeutic in use.The invention also contemplates continuous or discontinuousadministration, or daily doses divided into several partialadministrations. An appropriate cycle duration for a specific cancertherapeutic will be appreciated by the skilled artisan, and theinvention contemplates the continued assessment of optimal treatmentschedules for each cancer therapeutic. Specific guidelines for theskilled artisan are known in the art. See, e.g., Therasse et al., 2000,“New guidelines to evaluate the response to treatment in solid tumors.European Organization for Research and Treatment of Cancer, NationalCancer Institute of the United States, National Cancer Institute ofCanada,” J Natl Cancer Inst. February 2; 92(3):205-16.

Alternatively, longer treatment cycles may be desired. Accordingly, thecycle duration may range from approximately 10 to 56, 12 to 48, 14 to28, 16 to 24, or 18 to 20 days. The cycle duration may vary according tothe specific cancer therapeutic in use.

The present invention contemplates at least one cycle, preferably morethan one cycle during which a single cancer therapeutic or series oftherapeutics is administered. An appropriate total number of cycles, andthe interval between cycles, will be appreciated by the skilled artisan.The number of cycles may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or 21 cycles. The interval between cyclesmay be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or 21 days. The invention contemplates the continued assessmentof optimal treatment schedules for each cytotoxin and additional cancertherapeutic.

In another embodiment, a process is provided for preparing apharmaceutical for treating a mammal with cancer comprising the steps ofidentifying T-cell epitopes of bouganin having reduced propensity foractivated T-cells; preparing a cytotoxin of the invention having one ormore of the T-cell epitopes and suspending the protein in apharmaceutically acceptable carrier, diluent or excipient.

The invention also provides a pharmaceutical composition for treating amammal with cancer comprising a cytotoxin of the invention and apharmaceutically acceptable carrier, diluent or excipient.

The cytotoxins of the invention may be formulated into pharmaceuticalcompositions for administration to subjects in a biologically compatibleform suitable for administration in vivo. By “biologically compatibleform suitable for administration in vivo” is meant a form of thesubstance to be administered in which any toxic effects are outweighedby the therapeutic effects. The substances may be administered to livingorganisms including humans, and animals. Administration of atherapeutically active amount of the pharmaceutical compositions of thepresent invention is defined as an amount effective, at dosages and forperiods of time necessary to achieve the desired result. For example, atherapeutically active amount of a substance may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of antibody to elicit a desired response inthe individual. Dosage regime may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The active substance may be administered in a convenient manner such asby injection (subcutaneous, intravenous, intramuscular, etc.), oraladministration, inhalation, transdermal administration (such as topicalcream or ointment, etc.), or suppository applications. Depending on theroute of administration, the active substance may be coated in amaterial to protect the compound from the action of enzymes, acids andother natural conditions which may inactivate the compound.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985). On thisbasis, the compositions include, albeit not exclusively, solutions ofthe substances in association with one or more pharmaceuticallyacceptable vehicles or diluents, and contained in buffered solutionswith a suitable pH and iso-osmotic with the physiological fluids.

The pharmaceutical compositions may be used in methods for treatinganimals, including mammals, preferably humans, with cancer. It isanticipated that the compositions will be particularly useful fortreating patients with colorectal cancer, breast cancer, ovarian cancer,pancreatic cancer, head and neck cancer, bladder cancer,gastrointestinal cancer, prostate cancer, small cell and non small celllung cancer, sarcomas, gliomas, T- and B-cell lymphomas. The dosage andtype of cytotoxin to be administered will depend on a variety of factorswhich may be readily monitored in human subjects. Such factors includethe etiology and severity (grade and stage) of neoplasia.

Pharmaceutical compositions adapted for direct administration include,without limitation, lyophilized powders or aqueous or non-aqueoussterile injectable solutions or suspensions, which may further containantioxidants, buffers, bacteriostats and solutes that render thecompositions substantially isotonic with the blood of an intendedrecipient. Other components that may be present in such compositionsinclude water, alcohols, polyols, glycerin and vegetable oils, forexample. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets. Cytotoxin may besupplied, for example but not by way of limitation, as a lyophilizedpowder which is reconstituted with sterile water or saline prior toadministration to the patient.

Pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include essentially chemically inert and nontoxiccompositions that do not interfere with the effectiveness of thebiological activity of the pharmaceutical composition. Examples ofsuitable pharmaceutical carriers include, but are not limited to, water,saline solutions, glycerol solutions, ethanol,N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA),diolesyiphosphotidyl-ethanolamine (DOPE), and liposomes. Suchcompositions should contain a therapeutically effective amount of thecompound, together with a suitable amount of carrier so as to providethe form for direct administration to the patient.

In another embodiment, a pharmaceutical composition comprises acytotoxin and one or more additional cancer therapeutics, optionally ina pharmaceutically acceptable carrier.

The composition may be in the form of a pharmaceutically acceptable saltwhich includes, without limitation, those formed with free amino groupssuch as those derived from hydrochloric, phosphoric, acetic, oxalic,tartaric acids, etc., and those formed with free carboxyl groups such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In as far as this invention relates to modified bouganin, compositionscontaining such modified bouganin proteins or fragments of modifiedbouganin proteins and related compositions should be considered withinthe scope of the invention. A pertinent example in this respect could bedevelopment of peptide mediated tolerance induction strategies whereinone or more of the disclosed peptides is administered to a patient withimmunotherapeutic intent. Accordingly, synthetic peptides molecules, forexample one of more of comprising all or part of any of the epitoperegions R1-R3 as defined above. Such peptides are considered embodimentsof the invention.

In a further aspect of the present invention relates to methods fortherapeutic treatment of humans using the modified bouganincompositions. For administration to an individual, any of the modifiedcompositions would be produced to be preferably at least 80% pure andfree of pyrogens and other contaminants.

The present invention also provides a kit comprising an effective amountof a cytotoxin, optionally, in combination with one or more other cancertherapeutics, together with instructions for the use thereof to treatthe cancer.

(D) T-Cell Epitope Peptides

An additional embodiment of the invention is a T-cell epitope peptide.In an example, the T-cell epitope peptide is able to evoke a stimulationindex of greater than 1.8 in a T-cell assay, more preferably greaterthan 2.0. The T-cell epitope peptide of the invention is able to bindMHC class II.

In an embodiment of the invention the T-cell epitope peptide comprisesat least 9 consecutive amino acid residues from any of the sequences ofR1, R2 or R3 (above). In another embodiment, the T-cell epitope peptidesequence has greater than 90% amino acid identity with any one of thepeptide sequences R1, R2 or R3; more preferably the T-cell epitopepeptide has greater than 80% amino acid identity with any one of thepeptide sequences R1, R2 or R3.

The term “peptide” as used herein is a compound that includes two ormore amino acids. The amino acids are linked together by a peptide bond(defined herein below). There are 20 different naturally occurring aminoacids involved in the biological production of peptides, and any numberof them may be linked in any order to form a peptide chain or ring. Thenaturally occurring amino acids employed in the biological production ofpeptides all have the L-configuration. Synthetic peptides can beprepared employing conventional synthetic methods, utilizing L-aminoacids, D-amino acids, or various combinations of amino acids of the twodifferent configurations. Some peptides contain only a few amino acidunits. Short peptides, e.g., having less than ten amino acid units, aresometimes referred to as “oligopeptides”. Other peptides contain a largenumber of amino acid residues, e.g. up to 100 or more, and are referredto as “polypeptides”. By convention, a “polypeptide” may be consideredas any peptide chain containing three or more amino acids, whereas an“oligopeptide” is usually considered as a particular type of “short”polypeptide. Thus, as used herein, it is understood that any referenceto a “polypeptide” also includes an oligopeptide. Further, any referenceto a “peptide” includes polypeptides, oligopeptides, and proteins. Eachdifferent arrangement of amino acids forms different polypeptides orproteins. The number of polypeptides—and hence the number of differentproteins—that can be formed is practically unlimited.

Another embodiment of the invention is the use of the T-cell epitopepeptides of the invention to make the modified bouganin proteins of theinvention and modified T-cell epitope peptides.

A further embodiment of the invention is a modified T-cell epitopepeptide that is modified such that the modified T-cell epitope peptidehas reduced propensity to activate human T cells than the non-modifiedT-cell epitope peptide. In an example, the modified T-cell epitopepeptides of the invention contains modifications such that when testedin a T-cell assay evokes a reduced stimulation index in comparison tothe non-modified T-cell epitope peptide.

In an embodiment of the invention the modified T-cell epitope peptidehas the following sequence:

AKX ¹DRKX ²LX ³LGVX ⁴KL

wherein at least one of X¹, X², X³, and X⁴ is modified from thenon-modified sequence, as follows:

X¹ is T or A or Q;

X² is G or A;

X³ is Q or G; and

X⁴ is N or D or T or A or R or Q or E or G or H or K or S (SEQ ID NO:8).

In another embodiment of the invention the modified T-cell epitopepeptide has the following sequence:

LGVX ⁴KLEFSIEAIHG

wherein X⁴ is N or D or T or A or R or Q or E or G or H or K or S (SEQID NO:9).

In a further embodiment of the invention the modified T-cell epitopepeptide has the following sequence:

NGQEX ⁵AKFFLIVIQM

wherein X⁵ is Q or A (SEQ ID NO:10).

The invention also provides nucleic acid molecules encoding the T-cellepitope peptides or modified T-cell epitope peptides of the invention.

The following figures, sequence listings and examples are provided toaid the understanding of the present invention. It is understood thatmodifications can be made in the procedures set forth without departingfrom the spirit of the invention.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1 Method of Mapping Epitopes in Bouganin Using NaïveHuman T-Cell Proliferation Assays

Peptides covering the sequence of the mature bouganin protein, asdescribed by Den Hartog et al [ibid] were synthesized. The length ofeach peptide is 15 amino acids, and successive peptides overlap by 12residues. The sequence of these peptides and their numbering isindicated in TABLE 1.

The peptides were used in T-cell proliferation assays with PBMCs(peripheral blood mononuclear cells) from naïve donors (i.e. no knownsensitization to bouganin). 20 donor PBMC were selected to get anoptimal coverage of MHC class II allotypes. The allotypic coverage is inexcess of 85%. The HLA-DR allotypes are shown in TABLE 2.

PBMCs were stimulated with individual peptides in triplicate culturesfor 7 days before proliferation was assessed by ³H-thymidine (3H-Thy)incorporation. All peptides were tested at two different concentrations(1 μM and 5 μM). Stimulation indices (S.I.) were calculated as theamount of ³H incorporated, divided by the amount of ³H incorporated inmock-stimulated control cells.

Buffy coats from human blood stored for less than 12 hours were obtainedfrom the National Blood Service (Addenbrooks Hospital, Cambridge, UK).Ficoll-paque was obtained from Amersham Pharmacia Biotech (Amersham,UK). Serum free AIM V media for the culture of primary human lymphocytesand containing L-glutamine, 50 μg/ml streptomycin, 10 μg/ml gentomycinand 0.1% human serum albumin was from Gibco-BRL (Paisley, UK). Syntheticpeptides were obtained from Eurosequence (Groningen, The Netherlands)and Babraham Technix (Cambridge, UK).

Erythrocytes and leukocytes were separated from plasma and platelets bygentle centrifugation of buffy coats. The top phase (containing plasmaand platelets) was removed and discarded. Erythrocytes and leukocyteswere diluted 1:1 in phosphate buffered saline (PBS) before layering onto15 ml ficoll-paque (Amersham Pharmacia, Amersham UK). Centrifugation wasdone according to the manufacturers recommended conditions and PBMCswere harvested from the serum+PBS/ficoll paque interface. PBMCs weremixed with PBS (1:1) and collected by centrifugation. The supernatantwas removed and discarded and the PBMC pellet resuspended in 50 ml PBS.Cells were again pelleted by centrifugation and the PBS supernatantdiscarded. Cells were resuspended using 50 ml AIM V media and at thispoint counted and viability assessed using trypan blue dye exclusion.Cells were again collected by centrifugation and the supernatantdiscarded. Cells were resuspended for cryogenic storage at a density of3×10⁷ per ml. The storage medium was 90% (v/v) heat inactivated AB humanserum (Sigma, Poole, UK) and 10% (v/v) DMSO (Sigma, Poole, UK). Cellswere transferred to a regulated freezing container (Sigma) and placed at−70° C. overnight. When required for use, cells were thawed rapidly in awater bath at 37° C. before transferring to 10 ml pre-warmed AIM Vmedium.

PBMC were stimulated with protein and peptide antigens in a 96 well flatbottom plate at a density of 2×10⁵ PBMC per well. PBMC were incubatedfor 7 days at 37° C. before pulsing with ³H-Thy (Amersham-Pharmacia,Amersham, UK). Two control peptides termed C-32 and C-49 that havepreviously been shown to be immunogenic and a potent whole proteinnon-recall antigen Keyhole Limpet Hemocyanin (KLH) were used in eachdonor assay. C-32=sequence PKYVKQNTLKLAT from Flu haemagglutininresidues 307-319 (SEQ ID NO:127). C-49=sequence KVVDQIKKISKPVQH fromChlamydia HSP 60 (SEQ ID NO:128).

Peptides were dissolved in DMSO to a final concentration of 10 mM, thesestock solutions were then diluted 1/500 in AIM V media (finalconcentration 20 μM). Peptides were added to a flat bottom 96 well plateto give a final concentration of 1 and 5 μM in 100 μl. The viability ofthawed PBMC's was assessed by trypan blue dye exclusion, cells were thenresuspended at a density of 2×10⁶ cells/ml, and 100 μl (2×10⁵ PBMC/well)was transferred to each well containing peptides. Triplicate wellcultures were assayed at each peptide concentration. Plates wereincubated for 7 days in a humidified atmosphere of 5% CO² at 37° C.Cells were pulsed for 18-21 hours with 1 μCi ³H-Thy/well beforeharvesting onto filter mats. CPM values were determined using a Wallacmicroplate beta top plate counter (Perkin Elmer). Results were expressedas stimulation indices, derived by division of the proliferation score(e.g. counts per minute of radioactivity) measured to the test peptideby the score measured in cells not contacted with a test peptide.

Compilation of the results of the above assay indicates the presence offour T cell epitopes, corresponding to peptides 41, 44 and 50 in themature, processed region of the protein and peptide 88 in theunprocessed form. Since the epitope in peptide 88 is not part of themature protein, it is ignored under the scheme of the present invention.

For peptide 41 (termed epitope region R1) there were four responsivedonors to this peptide; donors 4, 5, 10 and 11. The S.I.s for these at 5μM are 3.6, 4.9, 2.1 and 2.0 respectively.

For peptide 44 (termed epitope region R2). There are two responsivedonors to this peptide; donors 4 (S.I.=3.5) and 11 (S.I.=2.3).Neighboring peptides 43 and 45 induced lower level T cell proliferationsince both these peptides overlap by 12 amino acids with peptide 44.

For peptide 50 there were 2 responsive donors to this peptide; donors 4(S.I.=2.9) and 14 (S.I.=2.0). Peptide 51 induced lower level T cellproliferation in donor 14 (S.I.>1.9).

The tissue types for all PBMC samples were assayed using a commerciallyavailable reagent system (Dynal, Wirral, UK). Assays were conducted inaccordance with the suppliers recommended protocols and standardancillary reagents and agarose electrophoresis systems. The allotypicspecificities of each of the responsive donor samples is given in TABLE2.

Example 2 Cloning of Bouganin from Bougainvillea spectabilis

Total RNA was extracted from the leaves of Bougainvillea spectabilisusing the ‘SV Total RNA Isolation System and protocols provided by thesupplier (Promega, Southampton, UK). Fresh leaf tissue was ground to afine powder under liquid nitrogen, and approximately 50 mg of groundtissue was used for the RNA isolation. RNA quality and quantity waschecked by visualization on a 1% agarose gel, and the bouganin gene wasamplified from the total RNA using the ‘Access RT-PCR System’ (Promega)using approximately 1 μg of RNA per reaction and with the gene specificprimers OL1032 and OL1033. Primer sequences are given in TABLE 3 below.This reaction generated a 1242 by fragment encompassing the nativeleader sequence and the full-length bouganin sequence. This fragment wascloned into the pGEM-T Easy vector (Promega), following kitinstructions, and designated pBou1. The sequence was confirmed by DNAsequencing.

The bouganin gene was transferred into the pET21a (Novagen, Nottingham,UK) by PCR cloning using the pBou1 plasmid as a template. A peIB(pectate lyase) leader sequence was added to the 5′ end, and a sequenceencoding a 6× histidine tag was added to the 3′ end of the bouganincoding sequence. The peIB leader was amplified from vector pPMI-his[Molloy, P. et al, (1995) J. Applied Bacteriology, 78: 359-365] usingprimer OL1322 (incorporating an Nde1 site) and primer OL1067. Thebouganin-his fragment was amplified from pBou1 using OL1068 and OL1323(incorporating a Not1 site). The peIB leader was fused in frame to thebouganin-his fragment using overlap PCR, and the resulting fragmentcloned into pGEM-T Easy (Promega). Following sequence confirmation thepeIB-bouganin-his fragment was cloned as a Nde1-Not1 fragment intoNde1-Not1 digested pET21a. This clone was designated pBou32.

Example 3 Construction of Mutant Bouganin Proteins

A number of modified (mutant) bouganin proteins were designed using dataprovided by the T-cell epitope mapping procedure and use of softwareable to simulate the binding of peptides with human MHC class II bindinggroove. This latter approach is described in detail elsewhere [WO02/069232]. Variant genes were constructed and the mutant proteinstested for functional activity. In general, “single mutant” proteinscontaining one amino acid substitution each were first constructed andtested, then genes for active modified proteins combined to producemultiply substituted modified proteins.

Mutant genes were constructed using an overlap PCR procedure in whichthe mutant amino acid codon becomes introduced into the gene by use of amutant in “overlap primer”. The scheme is well understood in the art andis described in detail elsewhere [Higuchi, et al (1900) Nucl. Acids Res.16:7351]. A total of 37 single mutant modified proteins were constructedand tested for retained functional activity. In addition, a negativecontrol modified protein containing a substitution Y70A was alsoconstructed and tested in all assays. One of the 37 “single mutant”modified proteins in fact contained two directly adjacent substitutions(E151T and 1152E) and is counted herein as a single mutant. Thesubstitutions tested and the corresponding activity values are given inTABLE 4.

A total of 11 multiple substitution modified proteins were constructedand tested for retained activity. The substitutions tested and thecorresponding activity values are given in TABLE 5.

TABLE 6 describes the sequences of the substitution modified proteins.TABLE 7 lists some specific sequences.

In all instances, proteins were purified and tested according to theprocedures outlined in examples 4 and 5 below.

Example 4 Expression of and Purification of Bouganin Protein

The plasmid pBou32 was transformed into BL21(DE3) (Novagen) competentcells following manufacturers instructions, and selected on LB(Invitrogen, Paisley, UK) plates containing 50 μg/ml carbenicillin. Afresh colony from this transformation was used to inoculate 5 ml 2xYT(Invitrogen) broth, without antibiotic, and this was grown with shakingat 250 rpm at 37° C. until OD600=1.5-2.0. The culture was thencentrifuged at 2500 rpm for 15 minutes at room temperature, and thecells resuspended in 5 ml fresh 2xYT plus 1 mM IPTG. This culture wasincubated at 30° C. with shaking at 300 rpm for 1.5 hours and the cellscollected by centrifugation and the supernatant discarded.

The cell pellet was resuspended in 1 ml of PEB2 (50 mM Tris-HCl'pH8, 20%sucrose, 1 mg/ml lysozyme, 1× Complete Protease Inhibitor Tablet (Roche,Lewes, UK), and incubated on ice for 1 hour with gentle mixing. The celldebris was centrifuged at 14,000 rpm at 4° C. and the pellet discarded.The resulting supernatant is now referred to as the ‘periplasmicfraction’. Bouganin protein was purified from the periplasmic fractionby nickel affinity column chromatography using commercially available“spin column” and the manufacturer's instructions (Qiagen, Crawley, UK).The resulting material was dialyzed against 4 liters of phosphatebuffered saline (0.138M NaCl, 0.0027M KCl, pH 7.4) overnight at 4° C.using a 10000 molecular weight cut-off ‘Slide-A-Lyzer’ (Pierce, Chester,UK). Following dialysis, the protein concentration was estimated usingthe Micro BCA Assay Kit (Pierce), and samples stored at −20° C.

Bouganin protein concentration was further determined using an ELISAbased assay system. Briefly, antiserum against bouganin was generated(Genovac, Freiburg, Germany), through the genetic immunization of tworats with a plasmid expressing bouganin. For the ELISA, recombinantbouganin is captured onto Ni-agarose coated plates via its His-tag andsubsequently detected with the rat antiserum and a secondaryHRP-conjugated anti-rat Fc antibody (Sigma, Poole, UK). As a standard, alarge preparation of the wild-type bouganin expressed in E. coli andquantitated using the total protein assay was used in eachdetermination.

Example 5 Assay of Bouganin Activity

The activity of the wild-type and modified (mutant) bouganin proteinswas tested by measuring their ability to inhibit protein synthesis in acell-free protein synthesis assay.

A mixture of 10 μl TNT Coupled Transcription/Translation mix (Promega),20 μM methionine, 120 ng pT7 luciferase DNA (Promega) and serialdilutions of WI and mutant bouganin protein in a final volume of 12.5 μlwere incubated at 30° C. for one hour, after which the reaction wasstopped by addition of 100 μl ‘SteadyGlow’ luciferase assay reagent(Promega). The luciferase activity was measured using a Wallacluminescence counter. Active bouganin protein is detected as a decreasein measured luciferase activity. Each modified bouganin protein wastested in at least 5 concentrations, with each data point in duplicate.Positive and negative controls were included in each experiment.

Results for single mutant proteins are shown in TABLE 4. Results formultiple mutant modified bouganin proteins are shown in TABLE 5. In eachinstance results are expressed relative to wild-type protein activity.All assays were conducted with the inclusion of an inactive mutantbouganin protein with a Y70A substitution.

In addition, luciferase assay results may be plotted showing %luciferase activity relative to control versus protein concentration ofadded bouganin. Examples of such plots are shown in FIG. 1 depicting theresults as determined for two different multiple mutant bouganinproteins.

Example 6 Assay of Variant Bouganin Sequences for Loss of T-CellEpitopes

The multiple modified protein designated Bou156 was selected for furthertesting using an immunogenicity assay. This variant contains thesubstitutions V123A, D127A, Y133N and 1152A. Immunogenicity testinginvolves use of live cells that may be damaged by testing using wholebouganin protein, therefore these assays were conducted using syntheticpeptides comprising the substitutions incorporated into variant Bou156.The peptides tested are listed in TABLE 8. The assays were conductedaccording to the procedures described in example 1 (above) using a PBMCdonor pool of 20 individuals. Peptides were tested in triplicate foreach donor sample at a two different final peptide concentrations (1 μMand 5 μM).

The results are expressed as SI per peptide per donor sample and areshown in FIG. 2. Del-41 is peptide sequence AKADRKALELGVNKL (SEQ IDNO:29). Del-44 is peptide sequence LGVNKLEFSIEAIHG (SEQ ID NO:30).Del-50 is peptide sequence NGQEAAKFFLIVIQM (SEQ ID NO:31). None of themodified peptides induced a T cell response in any of the donors(S.I.<2). In contrast an immunogenic control peptide stimulated T cellsof 6 donors (S.I.>2).

Example 7 VB6-845: Recombinant Engineering of an Ep-CAM-Specific FabAntibody for Optimal Delivery of De-Immunized Bouganin (De-Bouganin)

For this example and Example 8, the de-immunized bouganin used isBou156.

Tumor-targeting cytotoxins are composed of the variable region of anantibody linked to a bacterial, fungal or plant toxin. The present studyillustrates that the deimmunized bouganin constructs of the invention,comprising deimmunized bouganin linked to a targeting moiety havereduced immunogenicity, while still retaining their biological activity.TABLE 12 demonstrates the binding of the Ep-CAM antibody to severaltypes of tumours and thus shows that it can be used to treat these typesof cancers.

De-Immunized Bouganin Construct: Ep-CAM Directed Targeting Moiety Linkedto De-Bouganin

VB5-845, a Fab version of an anti-Ep-CAM scFv antibody, was geneticallylinked to a de-immunized form of bouganin (de-bouganin), Bou 156, apotent, plant-derived, type I ribosome-inactivating protein (RIP), tocreate the antibody-toxin construct VB6-845. FIG. 3 illustrates theconstruct VB6-845. FIG. 3A illustrates dicistronic unit of thepro-VB6-845, with peIB leader sequences. The amino acid sequence (SEQ IDNO:16) and nucleic acid coding sequence (SEQ ID NO:15) are provided inFIG. 3B. FIG. 3C illustrates the assembled VB6-845 protein, which isdescribed below in more detail. Testing of this construct, illustratethat the construct retained its biological activity (cytoxicity) and thespecificity of the targeting moiety (Ep-CAM antibody).

Orientation of the De-Immunized Bouganin Construct

To determine the optimal antibody-de-bouganin orientation, several formsof a dicistronic expression unit were generated, expressed and testedfor potency.

In each case, the dicistronic unit was cloned into the plNG3302 vector(FIG. 4) under the control of the arabinose-inducible araBAD promoterand transformed in E104 E. coli. Upon induction, the presence of thepeIB leader sequence directed the secretion of the Fab-de-bouganinfusion protein into the culture supernatant. The cleavable linkerenabled the de-bouganin to cleave from the targeting moiety and exertits biological activity. In one embodiment the linker is a furin linker,although a person skilled in the art would appreciate that othercleavable linkers could be suitable. Preferred linkers could be selectedbased on target specificity, and environment. A sample of the constructsmade and tested are as follows:

FIG. 3: VB6-845, wherein the de-bouganin (Bou156) is linked to theC-terminus of the CH domain via a furin linker. FIG. 3A illustrates thedicistronic unit of the pro-sequences, FIG. 3B illustrates the nucleicacid coding sequence (SEQ ID NO:15) and the amino acid sequence of thepro-sequences (SEQ ID NO:16) and FIG. 3C illustrates the assembledVB6-845 protein without the peIB sequences.

FIG. 5 illustrates the control Fab anti-Ep-CAM construct without theplant toxin, de-bouganin (VB5-845). FIG. 5A illustrates the dicistronicunit of the pro-sequences, FIG. 5B illustrates the nucleic acid codingsequence (SEQ ID NO:17) and the amino acid sequence of the pro-sequences(SEQ ID NO:18) and FIG. 5C illustrates the assembled VB6-845 proteinwithout the peIB sequences.

FIG. 6 illustrates the Fab anti-Ep-CAM de-bouganin construct,VB6-845-C_(L)-de-bouganin, wherein the Bou156 is linked at theC-terminus of the C_(L) domain. FIG. 6A illustrates the dicistronic unitof the pro-sequences, FIG. 6B illustrates the nucleic acid codingsequence (SEQ ID NO:19) and the amino acid sequence of the pro-sequences(SEQ ID NO:20) and FIG. 6C illustrates the assembledVB6-845-C_(L)-de-bouganin protein without the peIB sequences.

FIG. 7 illustrates the Fab anti Ep-CAM, de-bouganin construct,VB6-845-NV_(H)-de-bouganin, wherein Bou156 is linked to the N terminusof the V_(H) domain. FIG. 7A illustrates the dicistronic units of thepro-sequences, FIG. 7B illustrates the nucleic acid coding sequence (SEQID NO:21) and the amino acid sequence of the pro-sequences (SEQ IDNO:22) and FIG. 7C illustrates the assembled VB6-845-NV_(H)-de-bouganinprotein without the peIB sequences.

FIG. 8 illustrates the Fab anti-Ep-CAM constructVB6-845-NV_(L)-de-bouganin, wherein Bou156 is linked to the N-terminusof the V_(L) domain. FIG. 8A illustrates the dicistronic unit of thepro-sequences, FIG. 8B illustrates the nucleic acid coding sequence (SEQID NO:23) and the amino acid sequence of the pro-sequences (SEQ IDNO:24) and FIG. 8C illustrates the assembled VB6-845-NV_(L)-de-bouganinprotein without the peIB sequences.

In one embodiment, the de-bouganin molecule is linked to the C-terminalend of the heavy or light chains. The optimal configuration comprised apeIB leader sequence adjacent to V_(H)-C_(H) domain with an N-terminalhistidine affinity tag as the first unit. Immediately following was thesecond unit comprising the peIB-V_(L)-C_(L) domain linked to de-bouganinby a protease-sensitive linker. (FIG. 6) For constructs wherede-bouganin was re-positioned to the N-terminal end, Western-blotanalysis showed no detectable product and only C-terminal linkedde-bouganin (constructs of FIGS. 3 and 6) yielded an intact solubleprotein (FIG. 9), with good binding properties to Ep-CAM-positive celllines, as illustrated in the reactivity tests detected by flowcytometry. In the Western Blot analysis, FIG. 9 illustrates theexpression of VB6-845 and VB6-845 CL-de-bouganin in the supernatant ofinduced E104 cells at lab scale. An aliquot of the supernatant, 16microlitres, under non-reducing conditions, was loaded on a SDS-PAGEacrylamide gel and analysed by Western Blot using either a rabbitpolyclonal anti-4D5 antibody, followed by a goat anti-rabbit (1/2000),or a goat anti-human Kappa-light chain-HRP antibody (1/1000), to confirmthe identity and size of the recombinant protein. The arrow indicatesthe full-length VB6-845 (construct of FIG. 3) and VB6-845-CL-de-bouganin(construct of FIG. 6). Western blotting of non-induced E104 culturesupernatant revealed no corresponding bands demonstrating thespecificity of the antibodies (not shown).

The results of the reactivity tests with VB6-845 (FIG. 3) andVB6-845-CL-de-bouganin (FIG. 6) to Ep-CAM positive cell lines CAL 27 andNIH:OVCAR-3 as compared to a control (Ep-CAM-negative cell line, A-375)is illustrated in FIG. 10A. The results were comparable to the samereactivity tests conducted with another anti-Ep-CAM construct,VB6-845-gelonin, wherein the de-bouganin is replaced with another planttoxin, gelonin (See FIG. 14C showing its amino acid sequence (SEQ IDNO:26) and nucleic acid sequence (SEQ ID NO:25) The results of thereactivity test with the gelonin construct are illustrated in FIG. 10B.The addition of a second de-bouganin domain in the molecule with theoptimal orientation did not yield product.

The flow cytometry tests were conducted by incubating the constructs orcontrol with 0.45×10⁶ cells for an hour on ice. After washing, cellsurface bound constructs were detected with a rabbit anti-bouganin (forFIG. 10A) or mouse anti-His tag (FIG. 10B) for an hour on ice. The cellswere washed and incubated with FITC-conjugated sheep anti-rabbit IgG(FIG. 10A) and FITC-conjugated sheep anti-mouse (IgG) (FIG. 10B) for 30minutes on ice. Subsequently the cells were washed, resuspended in PBS5% FCS containing propidium iodide for assessment of antibody binding byflow cytometry. No shift in median fluorescence was detected followingincubation with VB6-845 and VB6-845-CL-de-bouganin with A-375. Incontrast, a marked shift in median fluorescence was observed with Ep-CAMpositive cell lines, CAL 27 and NIH:OVCAR-3 (FIG. 10A). As stated above,the results with VB6-845 were similar with the gelonin construct (FIG.10B).

ED-CAM Specificity

A competition assay of VB6-845 (construct of FIG. 3) with Proxinium™, ascFv format of VB6-845, but containing Pseudomonas exotoxin A,demonstrated that the Ep-CAM specificity of VB6-845 was unaltered whenengineered into a Fab format. (FIG. 11)

FIG. 11 illustrates the flow cytometry results of the competition assay,with VB6-845 at 1 and 10 μg/mL and increased concentration ofProxinium™, ranging from 0 to 100 μg/mL, were incubated with NIH:OVCAR-3cells (Ep-CAM positive tumour cell line). After 1 hour incubation at 4°C., cells were washed and bound VB6-845 was detected with a biotinylatedrabbit anti-bouganin followed by streptavidin-cychrome. The sameexperiment was performed with 4B5-PE which is used as a negativecontrol. The reaction conditions were as indicated on FIG. 11.

Potency (Biological Activity)

In addition, cell-free (FIG. 12) and MTS (FIGS. 13 A and B) assaysdemonstrated that de-bouganin retained its potency when conjugated tothe Fab fragment. The MTS assay used to measure potency was conductedusing standard technique known in the art, and as more fully describedbelow in Example 8. Using the Ep-CAM-positive cell lines, CAL 27 andNIH:OVCAR-3, the IC₅₀ of VB6-845 was 3 to 4 nM and 2 to 3 nM,respectively. In the case of VB6-845-C_(L)-de-bouganin, the potency wasmeasured at 1 to 2 nM for CAL 27 and 0.6 to 0.7 nM versus NIH:OVCAR-3.The development of Fab anti-Ep-CAM construct, comprising a human tumortargeting antibody fragment linked to a de-immunized bouganin shouldpermit repeat systemic administration of this drug and hence yieldgreater clinical benefit.

Harvesting of the Constructs

The constructs can be isolated from the cell cultures by techniquesknown in the art. For instance, if a His tag is placed at the N-terminalof the peptide construct, the Fab-bouganin protein can be purified usinga Ni²⁺-chelating capture method. As an example the following protocolcan be used. Conducting fed batch fermentation of VB6-845 variantsperformed in a 15 L CHEMAP fermenter using TB medium. At an OD₆₀₀ of 20(mid-log), the culture is induced with a mixture of feed and inducercontaining 50% glycerol and 200 g/l L-arabinose. At 30 hours postinduction, the culture is harvested, centrifuged at 8000 rpm for 30 minand VB6-845 variants purified using CM sepharose and Metal-ChargedChelating sepharose columns followed by a size exclusion column.Briefly, the supernatant is concentrated and diafiltered against 20 mMsodium phosphate pH 6.9±0.1. The diafiltered concentrated supernatant isthen applied onto a CM sepharose column equilibrated with 20 mM sodiumphosphate, 25 mM NaCl pH 6.9±0.1. The column is washed with 20 mM sodiumphosphate, 25 mM NaCl pH 6.9±0.1, bound VB6-845 is subsequently elutedwith 20 mM sodium phosphate, 150 mM NaCl pH 7.5±0.1. The CM sepharoseeluate is adjusted to contain a final concentration of 0.25% Triton-X100and applied to a charged chelating sepharose column. The chelatingsepharose column is then washed with 3 different wash buffers startingwith 20 mM sodium phosphate, 150 mM NaCl, 0.25% triton-X100 pH 7.5±0.1followed by 20 mM sodium phosphate, 150 mM NaCl pH 7.5±0.1 and followedby 20 mM sodium phosphate, 150 mM NaCl, 10 mM imidazole pH 7.5±0.1. Thebound VB6-845 is then eluted with 20 mM sodium phosphate, 150 mM NaCl,250 mM imidazole pH 7.5±0.1 and collected in 2 mL fractions. Theabsorbance at A₂₈₀ is determined for each fraction and the fractionswith material pooled are applied onto a size exclusion column S200 inorder to obtain a purity of >80%. In one embodiment, to increase theprotein purity and remove endotoxin, the pooled SEC fraction is diluted5-fold with 20 mM NaPO₄, pH 7.5 and passed though a Q-sepharose 15 mlfast flow column equilibrated with 20 mM NaPO₄, 25 mM NaCl pH 7.5 at aflow rate of about 5 ml/min. After application of the sample through thecolumn, the column is washed with 10 CV of equilibration buffer and thewash is pooled with the initial Q-sepharose flow through. The effluentis concentrated to ˜10-fold through the use of a 30 kDa MWCO membrane(Sartorius hydrosart membrane] to achieve a final concentration of 7.5mg/ml. Tween-80 is then added to a final concentration of 0.1%. Thefinal product is sterile filtered and stored at −80° C. Samples at eachsteps of the process are analyzed by Western blot after immunoblottingwith the anti-4D5 antibody. Purity is confirmed by colloidal bluestaining. The level of expression of VB6-845 variants is determined byWestern Blot analysis and ELISA.

Example 8 Functional and Biological Characterization of VB6-845, aRecombinant Ep-CAM-Specific Fab Antibody Genetically-Linked WithDe-Immunized Bouganin (De-Bouganin)

Chemotherapeutics are highly cytotoxic agents that often represent thestandard of care in the treatment of many of the solid tumor cancers.The cytotoxic action of these drugs targets rapidly dividing cells, bothnormal and tumor, thus creating a variety of adverse clinicalside-effects. VB6-845 is a Fab antibody linked to a de-immunized form ofthe plant-derived toxin bouganin. Unlike chemotherapeutics which lackdefined tumor-target specificity, VB6-845 restricts its cytolytic effectto Ep-CAM-positive tumor targets alone. In this study, flow cytometryanalysis and cytotoxicity were measured to assess the potency andselectivity of VB6-845.

Flow Cytometry

The tumour cell lines used in this study were purchase from ATCC andwere propagated following ATCC's recommendations except for the celllines C-4I, TOV-112D which were grown in RPMI 1640 or DMEM supplementedwith 10% FCS, respectively. Tumor cells were harvested at 60-70%confluence with viability over 90%. The human normal mammary epithelialcells (HMEC) were purchased from CAMBREX and maintained in specifiedmedia according to the procedure provided by CAMBREX. The cells wereharvested at 70% confluence with viability over 90%.

The gynaecological cell lines from endometrial ovarian and cervicalcancer indications were tested for VB6-845 binding on flow cytometry(Table 9). Ten microgram/mL of VB6-845 was added to each cell line(3×10⁵ cells) and incubated for 2 h at 4° C. A-375 and CAL 27 were usedas negative and positive cell line controls, respectively. After washingoff the unbound material, a mouse monoclonal anti-Histidine antibody(Amersham Pharmacia, Cat #27471001) diluted 1/800 in PBS containing 10%FCS was added and incubated for a further 1 hr at 4° C. Subsequently,FITC-labeled anti-mouse IgG (The Binding Site, Cat# AF271) diluted 1/100in PBS-10% FCS was added and incubated for 30 min. at 4° C. Finally, thecells were analyzed on a FACS Calibur following propidium iodidestaining to gate out the dead cells.

Cytotoxicity

The level of killing for VB6-845 in the cells listed in the flowcytometry study is as indicated in Table 10, indicated that theconstruct retained its de-bouganin cytotoxicity activity againstEp-CAM-positive cell lines. The cytoxicity was comparable to another FabVB6-845 variant containing a different plant-derived toxin, gelonin.(FIG. 14) FIG. 14 A compares the cytotoxicity of gelonin, Fabanti-Ep-CAM-gelonin construct (VB6-845-Gelonin) and the Fabanti-Ep-CAM-de-bouganin (Bou156) construct (VB6-845) in CAL 27 (FIG.14A) and NIH:OVCAR-3 cells (FIG. 14B). The nucleic acid and amino acidsequence of the VB6-845-gelonin construct is illustrate in FIG. 14C.

To study the specificity and selectivity of VB6-845 (Construct of FIG.3), the cytotoxic activity of VB6-845 (90% pure) was tested againstEp-CAM-positive (NIH:OVCAR-3) and Ep-CAM-negative (HMEC, DAUDI, A-375)cell lines (Table 11) along with 17 chemotherapeutic drugs (LKBLaboratories Inc.).

The MTS assay was preformed using standard techniques known in the art.More particularly, 50 microlitres of cells (2×10⁴ cells/ml) were seededper well and plates were incubated at 37° C. under 5% CO₂ for 2 hr. Then50 microlitres of spiked drug (i.e. construct to be tested or control)was added to the culture medium at increasing concentrations. Culturemedium, with or without cells, was used as positive and negativecontrols, respectively. The plates were left at 37° C. under 5% CO₂ for5 days. At day 5, the inhibition of cell proliferation was evaluated byadding 20 microlitres of MTS reagent (Promega, Cat# G5430). The plateswere further incubated at 37° C. under 5% CO₂ for 2 hr and ODs were readat 490 nm using the plate reader spetrophotometer. Background valueswere subtracted from the sample values obtained for each concentrationand the results were expressed as a percent of viable cells. The IC₅₀values for each drug were calculated for each cell line.

When assayed for cytotoxicity against NIH:OVCAR-3, an Ep-CAM-positiveovarian carcinoma, using a panel of standard chemotherapeutic agents,VB6-845 was shown to be more potent than 12 of the 17 drugs tested.(Table 11) Though 5 chemotherapeutics were more cytotoxic, they werealso shown to be far more toxic in that they lacked any cell-specifickilling. Of the five recommended chemotherapeutic agents for thetreatment of ovarian cancer (Paclitaxel, Carboplatin, Cisplatin,Doxorubicin and Topotecan), only two (Paclitaxel and Topotecan) weremore cytotoxic. While VB6-845 demonstrated highly potent cytolyticactivity in the range of 1 to 2 nM, the potent killing was restrictedexclusively to the Ep-CAM-positive tumor cell line NIH:OVCAR-3. Althoughsome killing of Ep-CAM-negative cell lines was exhibited with VB6-845,the cytotoxic effect was at least 220-fold and at most >1000-fold lesstoxic. VB6-845 thus represents a potent antibody-directed treatmentalternative to chemotherapeutics that when combined with the lowertoxicity profile, holds much promise in the treatment of many differenttypes of solid tumors.

Example 9 VB6-011: Recombinant Engineering of a Tumor-AssociatedAntigen-Specific Fab Antibody for Optimal Delivery of De-ImmunizedBouganin (De-Bouganin)

Tumor-targeting cytotoxins are composed of the variable region of anantibody linked to a bacterial, fungal or plant toxin. The present studyillustrates that the deimmunized bouganin constructs of the invention,comprising deimmunized bouganin linked to a targeting moiety havereduced immunogenicity, while still retaining their biological activity.TABLE 13 demonstrates the binding of the tumor-associated antigenantibody to several types of tumours and thus shows that it can be usedto treat these types of cancers.

De-Immunized Bouganin Construct: Tumor-Associated Antigen DirectedTargeting Moiety Linked to De-Bouganin

The H11 antibody, a monoclonal antibody recognizing tumor-associatedantigen, was genetically linked to a de-immunized form of bouganin(de-bouganin), Bou 156, a potent, plant-derived, type Iribosome-inactivating protein (RIP), to create the antibody-toxinconstruct VB6-011. FIG. 15 illustrates the nucleic acid coding sequenceand amino acid sequence. Testing of this construct, illustrates that theconstruct retained its biological activity (cytoxicity).

Potency (Biological Activity)

MTS assay demonstrated that de-bouganin retained its potency whenconjugated to the Fab fragment (FIG. 16). The MTS assay used to measurepotency was conducted using standard technique known in the art, and asmore fully described in Example 8.

Cytotoxicity

To study the specificity and selectivity of VB6-011, the cytotoxicactivity was tested against MB-435S cells. The MTS assay was performedusing standard techniques known in the art. More particularly, 50microlitres of cells (2×10⁴ cells/ml) were seeded per well and plateswere incubated at 37° C. under 5% CO₂ for 2 hr. Then 50 microlitres ofspiked drug (i.e. construct to be tested or control) was added to theculture medium at increasing concentrations. Culture medium, with orwithout cells, was used as positive and negative controls, respectively.The plates were left at 37° C. under 5% CO₂ for 5 days. At day 5, theinhibition of cell proliferation was evaluated by adding 20 microlitresof MTS reagent (Promega, Cat# G5430). The plates were further incubatedat 37° C. under 5% CO₂ for 2 hr and ODs were read at 490 nm using theplate reader spectrophotometer. Background values were subtracted fromthe sample values obtained for each concentration and the results wereexpressed as a percent of viable cells. Results show that the IC₅₀ valueof VB6-011 is 350 nM.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Position of Peptide first amino SEQ ID # acid NO Sequence 1 1 32YNTVSFNLGEAYEYP 2 4 33 VSFNLGEAYEYPTFI 3 7 34 NLGEAYEYPTFIQDL 4 10 35EAYEYPTFIQDLRNE 5 13 36 EYPTFIQDLRNELAK 6 16 37 TFIQDLRNELAKGTP 7 19 38QDLRNELAKGTPVCQ 8 22 39 RNELAKGTPVCQLPV 9 25 40 LAKGTPVCQLPVTLQ 10 28 41GTPVCQLPVTLQTIA 11 31 42 VCQLPVTLQTIADDK 12 34 43 LPVTLQTIADDKRFV 13 3744 TLQTIADDKRFVLVD 14 40 45 TIADDKRFVLVDITT 15 43 46 DDKRFVLVDITTTSK 1646 47 RFVLVDITTTSKKTV 17 49 48 LVDITTTSKKTVKVA 18 52 49 ITTTSKKTVKVAIDV19 55 50 TSKKTVKVAIDVTDV 20 58 51 KTVKVAIDVTDVYVV 21 61 52KVAIDVTDVYVVGYQ 22 64 53 IDVTDVYVVGYQDKW 23 67 54 TDVYVVGYQDKWDGK 24 7055 YVVGYQDKWDGKDRA 25 73 56 GYQDKWDGKDRAVFL 26 76 57 DKWDGKDRAVFLDKV 2779 58 DGKDRAVFLDKVPTV 28 82 59 DRAVFLDKVPTVATS 29 85 60 VFLDKVPTVATSKLF30 88 61 DKVPTVATSKLFPGV 31 91 62 PTVATSKLFPGVTNR 32 94 63ATSKLFPGVTNRVTL 33 97 64 KLFPGVTNRVTLTFD 34 100 65 PGVTNRVTLTFDGSY 35103 66 TNRVTLTFDGSYQKL 36 106 67 VTLTFDGSYQKLVNA 37 109 68TFDGSYQKLVNAAKV 38 112 69 GSYQKLVNAAKVDRK 39 115 70 QKLVNAAKVDRKDLE 40118 71 VNAAKVDRKDLELGV 41 121 72 AKVDRKDLELGVYKL 42 124 73DRKDLELGVYKLEFS 43 127 74 DLELGVYKLEFSIEA 44 130 75 LGVYKLEFSIEAIHG 45133 76 YKLEFSIEAIHGKTI 46 136 77 EFSIEAIHGKTINGQ 47 139 78IEAIHGKTINGQEIA 48 142 79 IHGKTINGQEIAKFF 49 145 80 KTINGQEIAKFFLIV 50148 81 NGQEIAKFFLIVIQM 51 151 82 EIAKFFLIVIQMVSE 52 154 83KFFLIVIQMVSEAAR 53 157 84 LIVIQMVSEAARFKY 54 160 85 IQMVSEAARFKYIET 55163 86 VSEAARFKYIETEVV 56 166 87 AARFKYIETEVVDRG 57 169 88FKYIETEVVDRGLYG 58 172 89 IETEVVDRGLYGSFK 59 175 90 EVVDRGLYGSFKPNF 60178 91 DRGLYGSFKPNFKVL 61 181 92 LYGSFKPNFKVLNLE 62 184 93SFKPNFKVLNLENNW 63 187 94 PNFKVLNLENNWGDI 64 190 95 KVLNLENNWGDISDA 65193 96 NLENNWGDISDAIHK 66 196 97 NNWGDISDAIHKSSP 67 199 98GDISDAIHKSSPQCT 68 202 99 SDAIHKSSPQCTTIN 69 205 100 IHKSSPQCTTINPAL 70208 101 SSPQCTTINPALQLI 71 211 102 QCTTINPALQLISPS 72 214 103TINPALQLISPSNDP 73 217 104 PALQLISPSNDPWVV 74 220 105 QLISPSNDPWVVNKV 75223 106 SPSNDPWVVNKVSQI 76 226 107 NDPWVVNKVSQISPD 77 229 108WVVNKVSQISPDMGI 78 232 109 NKVSQISPDMGILKF 79 235 110 SQISPDMGILKFKSS 80238 111 SPDMGILKFKSSKLT 81 240 112 MGILKFKSSKLTQFA 82 243 113LKFKSSKLTQFATMI 83 246 114 KSSKLTQFATMIRSA 84 249 115 KLTQFATMIRSAIVE 85252 116 QFATMIRSAIVEDLD 86 255 117 TMIRSAIVEDLDGDE 87 258 118RSAIVEDLDGDELEI 88 261 119 IVEDLDGDELEILEP 89 264 120 DLDGDELEILEPNIABouganin sequence peptides. The underlined residues are not present inthe mature protein

TABLE 2 Donor Donor No storage code Allotype 1 BC63 DRB1*04, DRB1*07,DRB4*01 2 BC86 DRB1*04, DRB1*15, DRB5 3 BC90 DRB1*07, DRB1*15, DRB4*01,DRB5 4 BC134 DRB1*01, DRB1*03, DRB3 5 BC167 DRB1*01, DRB1*07 and DRB4*016 BC216 DRB1*14, DRB1*15, DRB3, DRB5 7 BC217 DRB1*04, DRB1*12, DRB3,DRB4*01 8 BC233 DRB1*04, DRB1*11 and DRB3, DRB4*01 9 BC241 DRB1*07,DRB1*11, DRB3, DRB4*01 10 BC246 DRB1*01, DRB1*13 and DRB3 11 BC262DRB1*03, DRB1*07, DRB3, DRB4*01 12 BC292 DRB1*07, DRB1*13, DRB3, DRB4*0113 BC293 DRB1*04, DRB1*10, DRB4*01 14 BC231 DRB1*03 or DRB1*03, DRB1*13and DRB3 15 BC301 DRB1*07, DRB1*14, DRB3 16 BC326 DRB1*03, DRB1*15,DRB3, DRB5 17 BC316 DRB1*13, DRB1*15, DRB3, DRB5 18 BC321 DRB1*01,DRB1*15, DRB5 19 BC382 DRB1*04, DRB1*08, DRB4*01 20 BC336 DRB1*01,DRB1*11, DRB3 MHC Allotypes of PBMC donors

TABLE 3 SEQ ID Primer NO Sequence 0L1032 121 CATTACAAACGTCTACCAAGTTTOL1033 122 TTACAAAAGTAGATAAGTAATGTG 0L1322 123GATATACATATGAAATACCTATTGCCTACG 0L1067 124TGACACAGTGTTGTACGCTGGTTGGGCAGCGAGTAA OL1068 125GCTGCCCAACCAGCGTACAACACTGTGTCATTTAAC OL1323 126CGAGTGCGGCCGCTCAATGGTGATGGTGATGGTGT Sequences of primers used in theconstruction of the WT bouganin gene

TABLE 4 Single substitution bouganin variants con- structed and tested.Nucleotide Activity in Clone Mutation Mutations luciferase assay* ID**Negative control Y70A TAT-GCT −− BouY70A Epitope Region R1 (peptide 41)V123T GTG-ACG +/− Bou2 V123A GTG-GCT ++ Bou3 V123D GTG-GAT −− — V123EGTG-GAA −− — V123G GTG-GGC −− — V123H GTG-CAC −− — V123K GTG-AAG −− —V123N GTG-AAC −− — V123P GTG-CCT −− — V123Q GTG-CAA ++ Bou4 V123RGTG-AGA −− — V123S GTG-TCA −− — D127G GAT-GGC ++ Bou5 D127A GAT-GCT ++Bou6 E129K GAA-AAG −− — E129R GAA-AGA −− — E129Q GAA-CAA +/− Bou7 E129GGAA-GGC ++ Bou8 Epitope Region R2 (peptide 44) Y133P TAC-CCC −− — Y133NTAC-AAC ++ Bou9 Y133T TAC-ACA ++ Bou10 Y133A TAC-GCT ++ Bou11 Y133RTAC-AGA ++ Bou12 Y133D TAC-GAT ++ Bou13 Y133E TAC-GAA +/− Bou14 Y133QTAC-CAA ++ Bou15 Y133G TAC-GGC ++ Bou16 Y133H TAC-CAC ++ Bou17 Y133KTAC-AAG ++ Bou18 Y133S TAC-TCA ++ Bou19 Epitope Region R3 (peptide 50)E151T I152E GAGATA-ACGGAA −− — I152Q ATA-CAA ++ Bou20 I152A ATA-GCA ++Bou21 I152E ATA-GAA −− — F155P TTC-CCA −− — F155H TTC-CAC −− — I158PATT-CCA −− — *Activity in Luciferase assay: ++ = same or higher than WTprotein. + = within 2-fold of WT activity. +/− = within 3-fold of WTactivity. −− = less than one-third of WT activity. WT = Wild-typeprotein. **Clone ID. Designations for functionally active variants only.

TABLE 5 Multiple substitution bouganin variants constructed and tested.Epitope Epitope Epitope Activity in Clone Region R1 Region R2 Region R3luciferase ID (peptide 41) (peptide 44) (peptide51) assay Bou143 V123QY133Q I152Q ++ Bou144 V123A Y133N I152A ++ Bou145 V123A Y133Q I152A ++Bou146 V123A D127G ++ Bou147 V123A D127A ++ Bou148 V123Q D127G ++ Bou149V123Q D127A ++ Bou150 V123Q E129G + Bou151 V123A E129G + Bou156 V123AD127A Y133N I152A ++ Bou157 V123A D127A Y133Q I152A ++ *Activity inLuciferase assay: ++ = same or higher than WT protein. + = within 2-foldof WT activity. +/− = within 3-fold of WT activity. −− = less thanone-third of WT activity. WT = Wild-type protein.

TABLE 6 Clone ID Substitution(s)* Protein Bou32 WT SEQ ID No 1 Bou156V123A, D127A, Y133N, I152A SEQ ID No 13 Bou157 V123A, D127A, Y133Q,I152A SEQ ID No 14 Bou143 V123Q, Y133Q, I152Q Bou144 V123A, Y133N, I152ABou145 V123A, Y133Q, I152A Bou146 V123A, D127G Bou147 V123A, D127ABou148 V123Q, D127G Bou149 V123Q, D127A Bou150 V123Q, E129G Bou151V123A, E129G Bou2 V123T Bou3 V123A Bou4 V123Q Bou5 D127G Bou6 D127A Bou7E129Q Bou8 E129G Bou9 Y133N Bou10 Y133T Bou11 Y133A Bou12 Y133R Bou13Y133D Bou14 Y133E Bou15 Y133Q Bou16 Y133G Bou17 Y133H Bou18 Y133K Bou19Y133S Bou20 I152Q Bou21 I152A *The numbering commences from residue 1 ofthe bouganin reading frame and therefore excludes a PelB leader sequenceincluded in most constructs.

TABLE 7 SEQ ID No 1 ProteinYNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKVDRKDLELGVYKLEFSIEAIHGKTINGQEIAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPDMGILKFKSSK SEQ ID No 13 ProteinYNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKADRKALELGVNKLEFSIEAIHGKTINGQEAAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPDMGILKFKSSK

TABLE 8 Modified and WT peptides of Bouganin further tested in T cellassays. Position of first SEQ Peptide amino acid within ID numberbouganin Sequence* NO DeI-41 121-135 AKADRKALELGVNKL 29 DeI-44 130-144LGVNKLEFSIEAIHG 30 DeI-50 149-163 NGQEAAKFFLIVIQM 31 *Substituted(mutant) residue underlined

TABLE 9 VB6-845 binding to gynecological cell lines by flow cytometryVB6-845 (fold increase Indication Cell lines MF ± SEM) EndometrialHEC-1-A 42.3 ± 0.9  RL95-2 4.9 ± 0.7 SK-UT-1 1.1 ± 0.1 Ovarian NIH:OVCAR-3 33.6 ± 6.0  SK-OV-3 4.3 ± 1.0 TOV-112G 1.1 ± 0.1 Cervical HT-329.1 ± 1.2  C-4 I 6.8 ± 0.6 C-33A 1.1 ± 0.0 Melanoma A-375 1.1 ± 0.1Results are expressed as fold-increase in MF ± SEM.

TABLE 10 VB6-845-mediated Cytotoxicity by MTS assay IC₅₀ nM VB6-845Indication Cell line 70% pure Endometrial HEC-1-A 43 KLE >100 RL95-2 100Ovarian NIH-OVCAR-3 3.4 Caov-3 1.3 SK-OV-3 >100 Cervical MS751 0.43 HT-323 ME-180 37 C-4 I 1.7 Melanoma A-375 >100

TABLE 11 Specificity and selectivity of VB6-845 Versus ChemotherapeuticsIC₅₀ nM NIH: OVCAR-3 A-375 DAUDI HMEC Paclitaxel <10⁻⁶ 4.9 × 10⁻⁶ <10⁻⁶<10⁻⁶ Docetaxel <10⁻⁶ <10⁻⁶ <10⁻⁶ <10⁻⁶ Vincristine 4.4 × 10⁻⁶ <10⁻⁶<10⁻⁶ <10⁻⁶ Vinblastine 1.1 × 10⁻⁶ <10⁻⁶ <10⁻⁶ <10⁻⁶ Sulfate Topotecan0.071 1.5 0.009 4.1 VB6-845 1 >1000 >1000 220 (90% pure) Doxorubicin 32.8 16 × 10⁻⁶ 16 Mitomycin C 28 14 2.8 50 Bleomycin 30 170 22 600Sulfate Bleomycin 150 290 130 1000 A5 Irinotecan 180 900 190 1000Etoposide 210 280 1.7 600 Methotrexate >1000 6 3.6 41Chloram- >1000 >1000 >1000 >1000 bucilFluorouracil >1000 >1000 >1000 >1000 Cyclo- >1000 >1000 >1000 >1000phosphamide Cisplatin >1000 >1000 >1000 >1000Carboplatin >1000 >1000 >1000 >1000

TABLE 12 VB6-845 Tumor Cell Indications Binding for scFv 845 INDICATIONSN¹ (IgG)² Gastric 3 148.9 Ovarian 2 84.1 Esophageal 3 72.4 Bladder 1459.6 Prostate 5 50.1 Cervical 3 37.5 Endometrial 1 23.8 Lung 3 16.4 Headand Neck 2 11.4 Kidney 3 9.4 Pancreas 3 5.5 Melanoma 3 1.6 ¹N indicatesthe number of cell lines tested per indication. ²Mean fold-increase inmedian fluorescence over the control antibody from all cell lines ineach indication.

TABLE 13 VB6-011 Tumor Cell Indications Binding for mAb 011 INDICATIONSN¹ (IgG)² Breast 3 16.9 Prostate 3 15.1 Melanoma 3 14.0 Lung 3 13.1Ovarian 2 11.1 Colon 3 8.7 Kidney 3 6.9 Liver 2 6.5 Pancreas 3 4.2 Headand Neck 2 2.9 ¹N indicates the number of cell lines tested perindication. ²Values indicate the mean calculated from the sum of themean fold increase in median fluorescence over the control antibody fromall cell lines in each indication. A zero value would mean no measurablereactivity relative to the control activity

1-17. (canceled)
 18. A method of inhibiting or destroying a cancer cellcomprising administering a cytotoxin to an animal in need thereof,wherein said cytotoxin comprises a targeting moiety attached to amodified bouganin protein, wherein said modified bouganin has a reducedpropensity to activate an immune response.
 19. The method of claim 18,wherein the cancer cell is selected from the group consisting ofcolorectal cancer, breast cancer, ovarian cancer, pancreatic cancer,head and neck cancer, bladder cancer, liver cancer, renal cancer,melanomas, gastrointestinal cancer, prostate cancer, small cell and nonsmall cell lung cancer, sarcomas, gliomas, T- and B-cell lymphomas. 20.A method of treating cancer comprising administering an effective amountof a cytotoxin to an animal in need thereof, wherein said cytotoxincomprises a targeting moiety attached to a modified bouganin protein,wherein said modified bouganin has a reduced propensity to activate animmune response.
 21. The method of claim 20, wherein the cancer isselected from the group consisting of colorectal cancer, breast cancer,ovarian cancer, pancreatic cancer, head and neck cancer, bladder cancer,liver cancer, renal cancer, melanomas, gastrointestinal cancer, prostatecancer, small cell and non small cell lung cancer, sarcomas, gliomas, T-and B-cell lymphomas.
 22. A pharmaceutical composition comprising thecytotoxin and a pharmaceutically acceptable carrier, diluent orexcipient, wherein said cytotoxin comprises a targeting moiety attachedto a modified bouganin protein, wherein said modified bouganin has areduced propensity to activate an immune response.
 23. A process ofpreparing a pharmaceutical for treating an animal with cancer comprising(a) identifying T-cell epitopes of bouganin; (b) modifying one or moreamino acid residues in a T-cell epitope to prepare a modified bouganinhaving reduced propensity to activate T-cells; (c) preparing a cytotoxinhaving a cancer-binding ligand attached to the modified bouganin; and(d) suspending the cytotoxin in a pharmaceutically acceptable carrier,diluent or excipient.
 24. The process of claim 23, wherein the cancer isselected from the group consisting of colorectal cancer, breast cancer,ovarian cancer, pancreatic cancer, head and neck cancer, bladder cancer,liver cancer, renal cancer, melanomas, gastrointestinal cancer, prostatecancer, small cell and non small cell lung cancer, sarcomas, gliomas, T-and B-cell lymphomas. 25-34. (canceled)